Method of treating dermatitis comprising administering a chymase inhibitor

ABSTRACT

A medicament safe and free from side effects, for the prevention or treatment of dermatitis exhibiting a biphasic inflammation reaction or dermatitis induced by repeated exposure to an antigen, which suppresses the progress of the condition and improves the quality of life of the patient, which medicament treats dermatitis exhibiting a biphasic inflammation reaction or dermatitis induced by repeated exposure to an antigen by including a quiazoline derivative having the formula (I) or (II) or its pharmaceutically acceptable salt as an effective ingredient:

TECHNICAL FIELD

The present invention relates to medical applications of a chymaseinhibitor, more particularly relates to a medicament for the preventionor treatment of dermatitis, which has a chymase inhibitor as aneffective ingredient.

BACKGROUND ART

Inflammatory reactions are caused by bacteria, viruses, and otherpathogens and by trauma, foreign matter, etc. They are immunoreactionswhere granulocytes, monocytes, lymphocytes, and other immune cells expelpathogens or injured tissues or foreign materials. Dermatitis is anacute or chronic inflammation of the skin, and in particular, there hasbeen a remarkable rise in atopic dermatitis in recent year, which isbecoming a major problem.

Atopic dermatitis is a chronic disease in which eczema with itch is themajor condition, with exacerbation and remission observed by turns. Inmost cases, the patient or his or her family has a history of cnidosisor allergic rhinitis and bronchial asthma and other allergic ailments(J. Allergy Clin. Immunol. 104, S123, 1999). The symptoms of atopicdermatitis are diverse and the causes are still unclear, but it isbelieved that the disease is elicted mainly by various naturalsubstances including ticks, hair, feathers, bacteria and mycetes, orfoods including eggs, milk, or synthetic products including chemicalfibers and detergents, etc. It is also pointed out that the disorder inthe barrier function of the skin due to dry skin plays an important rolein atopic dermatitis.

The mechanism of onset of atopic dermatitis is still not clear. It hasbeen thought that the type I allergic reaction (immediate allergicreaction), in which IgE and mast cells are involved, plays an importantrole in atopic dermatitis, since this disease is one of hypersensitivityreactions to a series of antigens, the patients or their familysometimes have other allergic disorders, and an increase in the serumIgE level is observed in many cases. However, anti-allergic agents thatinhibit type I allergic reaction are ineffective or exhibit notherapeutic effect in atopic dermatitis, showing that the involvement ofthe type I reaction in the pathogenesis of this disease is only partial.

It has been reported, on the other hand, that the patients with atopicdermatitis show biphasic skin reaction, when they are exposed to theallergens (J. Allergy Clin. Immunol. 101, 222, 1998). This biphasic skinreaction is, for example, observed in the case of intradermaladministration of an antigen such as Ascalis extract to animalssensitized to the same antigen (J. Immunol. 131, 1096, 1983). The firstreaction, termed early-phase reaction, peaks 1 hr after the antigenchallenge. The second reaction, late-phase reaction, is known to showthe maximal response after 8 to 24 hours (Biol. Pharm. Bull. 18, 239,1995). Early-phase reaction is suppressed by antagonists to histamineacceptors, suggesting that the reaction is induced by IgE and mastcells. In contrast, the mechanism of late-phase reaction is notnecessarily clear, but is characterized by a remarkable infiltration ofeosinophils in the skin (Int. Arch. Allergy Immunol. 113, 196, 1997),which is the typical histological feature observed in the patients withatopic dermatitis (J. Am. Acad. Dermatol. 24, 1101, 1991). Further, theseverity of atopic dermatitis patients is known to be correlated withthe serum level of ECP (eosinophil cationic proteins), and the number ofperipheral eosinophils (Medicina 34, 220, 1997). Further, in recentyears, it has been pointed out that the clinical symptoms of atopicdermatitis are extremely similar to the symptoms of contact dermatitisclassified as a type IV allergic reaction (Medicina 34, 220, 1997).These findings suggest the possibility that a type IV allergic reactionis also involved in the mechanism of pathogenesis of the disease.

It is generally known that contact hypersensitivity reaction, therepresentative type IV allergic reaction, is induced by applying haptensuch as DNFB (dinitrofluorobenzene) to the mice that had been sensitizedonce with the same hapten, but it has been recently reported that a typeI allergic reaction is induced in addition to a type IV allergicreaction when repeatedly applying such a hapten to the skin (J. Invest.Dermatol. 105, 749, 1995). For example, by repeatedly applying hapten,the IgE level in the blood rises and the time-course of the reactionshifts to that of type I allergic reaction, by repeating the haptenchallenge. In such an animal model, not only the transit response tohapten challenge, but also the baseline of skin thickness, the thicknessbefore the hapten challenge, gradually increases, which seems to be afeature of chronic dermatitis. These findings suggest that thedermatitis induced by repeating application of hapten is thought to beuseful as an animal model of atopic dermatitis (Anitex 10, 23, 1998).

Recently, it has been reported that NC/Nga mice spontaneously developdermatitis similar to atopic dermatitis (Int. Immunol. 9, 461, 1997).NC/Nga mice that are maintained in conventional, non specific pathogenfree environment begin to exhibit remarkable scratching behavior anderythema after about seven to eight weeks of age, then exhibithemorrhaging or sores or ulceration of the skin with aging. Further,they exhibit symptoms resembling the clinical observations of atopicdermatitis in humans such as drying or thickening of the skin. Otherstrains of mice such as BALB/c do not suffer from similar dermatitiseven if made to cohabitate with NC/Nga mice, suggesting that thisdermatitis is considered specific to NC/Nga mice (Saishin Igaku (LatestMedicine), 53, 2848, 1998). Further, when raising these mice underspecific paphogen free (SPF) environment, no skin abnormalities areobserved at all, raising the possibility that some sort of environmentalfactors are involved in the onset of dermatitis in these mice. WhenNC/Nga mice raised under an SPF environment are repeatedly painted withhapten, only the delayed type hypersensitivity reaction called contactdermatitis is caused in the initial period of sensitization, but withthe increase in sensitization, conditions similar to atopic dermatitisare observed (CRJ Letters 11, 1, 1998). Therefore, while the naturalstimulant for the spontaneous dermatitis in these mice is still notclear, it is clear that repeated exposure to some sort of antigen underthe natural environment is an important factor. Thus, these mice areextremely useful as a model for atopic dermatitis spontaneously causedby repeated exposure to an allergen that would be present in the air.

The most effective medicament for the treatment of atopic dermatitis isa steroid ointment (J. Allergy Clin. Immunol. 104, S123, 1999). Use ofsuch steroid ointment, however, requires careful selection of themedicament used according to the location of application and timing. Ifthe method of use is not appropriate, no effect will be manifested orthe condition will conversely deteriorate. Further, when a steroidointment is used over a long period, side effects such as atrophy androsacea occur. Further, if stopping use of this medicament mid way, thephenomenon of rebound, that is, remarkable deterioration of the skinsymptoms, is sometimes observed.

In addition to steroid ointment, histamine antagonists and anti-allergicagents have been used for treatment of atopic dermatitis. Histamineantagonists are effective in the sense of eliminating ichiness, but donot lead to a cure of this disease. Anti-allergic agents such astranilast, ketotifen, oxatomide, and azelastine hydrochloride areineffective against conditions of atopic dermatitis, or the effect islittle, if any. This is believed to be due to the fact that these drugshave a suppressive action on a type I allergic reaction, but exhibitalmost no effect on the actions of eosinophils or type IV allergicreaction (Jap. J. Pharmacol. 63, 73, 1993, Jap. J. Pharmacol. 51, 93,1989). Recently ointment of tacrolimus, an immunosuppressive, has beendeveloped as a medicament for treatment of atopic dermatitis (J. AllergyClin. Immunol. 104, S126, 1999), but various side effects due tosuppression of the immunoreaction by use of this medicament cannot beavoided. Taken together, it is difficult to say that any of the existingmedicaments are sufficiently satisfactory in respect to efficacy andside effects, and development of a medicament superior in efficacy andsafety is desirable.

On the other hand, chymase is a serine protease stored in mast cellgranules, and widely present in tissue such as the skin, heart, vascularwalls, intestines, etc. (Mast Cell Proteases in Immunology and Biology;Caughey, G. H., Ed; Marcel Dekker, Inc.; New York, 1995). It has beenreported long ago that chymase acts on rat peritoneal mast cells andcauses degranulation (J. Immunol. 136, 3812, 1986) and that a chymaseinhibitor suppresses the Ig-E demiated mast cell degranulation (Biochem.Int. 10, 863, 1985) and has been pointed out that chymase is involved inthe function of mast cells. Recently, it has been reported thatadministration of human chymase induces infiltation of leukocytesincluding eosinophils in mice as well as guinea pigs (Br. J. Pharmacol,125, 1491, 1998), that human chymase acts on the precursor of IL-1β(Interleukin 1β) and converts it to active type IL-1β (J. Exp. Med. 174,821, 1991), and that human chymase has the action of partially digestingmembrane-bound stem cell factor (SCF) and converting it to soluble SCF(Proc. Natl. Acad. Sci. U.S.A. 94, 9017, 1997), etc. These findingssuggest the possibility that chymase has some sort of role in allergicdiseases such as atopic dermatitis. However, it is difficult to say thatthe pathophysiological role of chymase has been elucidated by thesestudies. At the present time, an energetic search is going on forsubstances which can inhibit the activity of chymase in vivo with theaim of clarifying the role of chymase in various diseases and thepossibility of chymase inhibitors as pharmaceuticals.

There are chymase inhibitors such as low molecular weight chymaseinhibitors such as shown in textbooks (Protease Inhibitors; Barrett etal., Eds; Elssevier Science B. V.; Amsterdam, 1996), α-keto acidderivatives reported as peptide type inhibitors (WO93-25574, Proc. Natl.Acad. Sci. USA, 1995, 92, 6738), α-difluoro-β-keto acid derivatives(Japanese Unexamined Patent Publication (Kokai) No. 9-124691),tripeptide inhibitors (WO93-03625), phosphoric acid derivatives(Oleksyszyn et al., Biochemistry 30, 485, 1991), peptide like inhibitorssuch as trifluoromethylketone derivatives (WO96-33974, JapaneseUnexamined Patent Publication (Kokai) No. 10-53579) and acetoamidederivatives (Japanese Unexamined Patent Publication (Kokai) No. 10-7661,Japanese Unexamined Patent Publication (Kokai) No. 10-53579, JapaneseUnexamined Patent Publication (Kokai) No. 11-246437, WO99-41277,WO98-18794, WO96-39373), non-peptide type inhibitors such as triazinederivatives (Japanese Unexamined Patent Publication (Kokai) No. 8-208654and Japanese Unexamined Patent Publication (Kokai) No. 10-245384),phenol ester derivatives (Japanese Unexamined Patent Publication (Kokai)No. 10-87567), cephem derivatives (Japanese Unexamined PatentPublication (Kokai) No. 10-87493), isoxazole derivatives (JapaneseUnexamined Patent Publication (Kokai) No. 11-1479), imidazolidinederivatives (WO96-04248), hydantoin derivatives (Japanese UnexaminedPatent Publication (Kokai) No. 9-31061), quinazoline derivatives(WO97-11941), etc. have been reported, but no satisfactory medicament ortreatment method using inhibition of the activity of chymase as astrategy for treatment has yet been established.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a side effect-free,safe medicament for prevention or treatment of dermatitis such as atopicdermatitis, which exhibits biphasic skin reaction or is induced byrepeated exposure to an antigen, which suppresses the progress of thecondition and improves the quality of life of the patient.

The present inventors engaged in intensive studies taking note of thefact that atopic dermatitis exhibits biphasic skin reaction and thatlate-phase reaction plays an important role in the condition. As aresult, the present inventors discovered that a chymase inhibitor actsto alleviate the late-phase reaction in the biphasic skin reaction ofdermatitis and that it is effective even against dermatitis caused byrepeated exposure to an antigen, and thereby completed the presentinvention.

That is, according to the present invention, there is provided amedicament for prevention or treatment of dermatitis exhibiting biphasicskin reaction containing a chymase inhibitor as its effectiveingredient.

According to the present invention, further, there is provided amedicament for alleviation of late-phase reaction of dermatitisexhibiting biphasic skin reaction containing a chymase inhibitor as itseffective ingredient.

According to the present invention, further, there is provided amedicament for the prevention or treatment of dermatitis induced byrepeated exposure to an antigen containing a chymase inhibitor as itseffective ingredient.

According to the present invention, there is provided a pharmaceuticalcomposition for the prevention or treatment of dermatitis exhibitingbiphasic skin reaction containing a chymase inhibitor in an amountalleviating the late-phase reaction and a pharmaceutically acceptablevehicle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the time-course of the skin reaction inAscaris-induced mouse biphasic skin reaction in Example 2.

FIGS. 2A, 2B, and 2C are graphs showing the effects of chymase inhibitor(FIG. 2A) and control drugs, prednisolone (FIG. 2B) and diphenhydramine(FIG. 2C), in Ascaris-induced biphasic skin reaction in Example 3.

FIGS. 3A and 3B are graphs showing the time-course of the skin reactionwhen the human chymase (FIG. 3A) or histamine (FIG. 3B) was administeredintradermally to mice in Example 4.

FIGS. 4A and 4B are graphs showing dose-dependency of chymase in theskin reaction when human chymase was administered intradermally to micein Example 4 (FIG. 4A, 1 hour after the chymase administration, FIG. 4Bafter 16 hours after the chymase administration).

FIG. 5 is a graph showing the effect of heat treatment on the action ofhuman chymase on inducing dermatitis in Example 5.

FIGS. 6A to 6E are photos showing the histologically analyzing thedermatitis induced by intradermal injection of human chymase in Example6, wherein FIG. 6A, normal mice; FIG. 6B, dermatitis induced by Ascarisextract (after 1 hour); FIG. 6C, dermatitis induced by Ascaris extract(after 24 hours), FIG. 6D, dermatitis induced by human chymase (after 1hour); FIG. 6E dermatitis induced by human chymase (after 24 hours).

FIGS. 7A and 7B are graphs showing the skin reaction when human chymaseadministered intradermally to mast cell-deficient mice in Example 7(FIG. 7A, the reaction after 1 hour; FIG. 7B the reaction after 16hour).

FIG. 8A is a view showing the concentration dependency of the effect ofhuman chymase on migration of human polymorphonuclear leukocytes (PMN)in vitro in Example 8. FIG. 8B is a graph showing the effect of achymase inhibitor on the chymase-induced PMN migration in Example 8.

FIG. 9 is a graph showing the time-course of the increase in earthickness in the dermatitis induced by repeated application of hapten inExample 9.

FIG. 10 is a graph showing the change in chymase-like activity in theear in the dermatitis induced by repeated application of hapten inExample 9.

FIGS. 11A to 11D are views showing the effects of chymase inhibitor inthe dermatitis induced by repeated application of hapten in Example 10(FIG. 11A, control drug, prednisolone, FIG. 11B to FIG. 11D, Compound35, Compound 34, and Compound 18, respectively).

FIG. 12 is a graph showing the effect of a chymase inhibitor on theincrease in the number of eosinophils in the dermatitis induced byrepeated application of hapten in Example 11.

FIG. 13 is a graph showing the effect of a chymase inhibitor on theincrease in the number of mast cells in the dermatitis induced byrepeated application of hapten in Example 12.

FIGS. 14A, 14B, and 14C are representative photos showing the effect ofa chymase inhibitor on the increase in the number of eosinophils in thedermatitis induced by repeated application of hapten in Example 12.

FIG. 15 is a graph showing the time-course of skin thickness when humanchymase was repeatedly injected intradermally to mice in Example 13.

FIG. 16 is a graph showing the time-course of the number of eosinophilsin the skin when human chymase was repeatedly injected intradermally tomice in Example 4.

FIG. 17 is a graph showing the time-course of histamine content in theskin when human chymase was repeatedly injected intradermally to mice inExample 15.

FIGS. 18A and 18B are photos showing representative photos ofimmunohistological analysis for SCF expression when human chymase wasrepeatedly injected intradermally to mice in Example 16 (FIG. 18A,normal skin; FIG. 18B, chymase-injected)

FIG. 19 is a graph showing the effect of human chymase on SCF expressionin human keratinocytes in vitro in Example 17.

FIGS. 20A and 20B are graphs showing the effect of a chymase inhibitoron clinical skin scores in NC/Nga mice in Example 18. FIG. 20A, at thestart of the experiment;

FIG. 20B, 35 days after the start of the chymase administration.

FIG. 21 is a graph showing the effect of a chymase inhibitor onhistological scores in NC/Nga mice in Example 18.

FIGS. 22A and 22B are graphs showing the effect of a chymase inhibitoron the number of skin mast cells in NC/Nga mice in Example 18. FIG. 22A,the ear; FIG. 22B, the back.

FIGS. 23A and 23B are graphs showing the effect of a chymase inhibitoron the number of skin eosinophils in NC/Nga mice in Example 18 FIG. 23A,the ear; FIG. 23B, the back.

BEST MODE FOR CARRYING OUT THE INVENTION

The chymase inhibitor able to be used in the present invention can beselected as a substance able to exhibit an action inhibiting theactivity of chymase by the use of methods workable by persons skilled inthe art. As the method of selection, for example, the method of thelater explained Example 1 may be mentioned. The compounds obtained inthis way include known compounds previously reported as chymaseinhibitors, for example, the low molecular weight chymase inhibitorssuch as shown in textbooks (Protease Inhibitors; Barrett et al., Eds;Elssevier Science B. V.; Amsterdam, 1996), α-keto acid derivativesreported as peptide type inhibitors (WO93-25574, Proc. Natl. Acad. Sci.USA, 1995, 92, 6738), α,α-difluoro-β-keto acid derivatives (JapaneseUnexamined Patent Publication (Kokai) No. 9-124691), tripeptideinhibitors (WO93-03625), phosphoric acid derivatives (Oleksyszyn et al.,Biochemistry 30, 485, 1991), peptide like inhibitors such astrifluoromethylketone derivatives (WO96-33974, Japanese UnexaminedPatent Publication (Kokai) No. 10-53579) and acetoamide derivatives(Japanese Unexamined Patent Publication (Kokai) No. 10-7661, JapaneseUnexamined Patent Publication (Kokai) No. 10-53579, Japanese UnexaminedPatent Publication (Kokai) No. 11-246437, WO99-41277, WO98-18794,WO96-39373), non-peptide type inhibitors such as triazine derivatives(Japanese Unexamined Patent Publication (Kokai) No. 8-208654 andJapanese Unexamined Patent Publication (Kokai) No. 10-245384), phenolester derivatives (Japanese Unexamined Patent Publication (Kokai) No.10-87567), cephem derivatives (Japanese Unexamined Patent Publication(Kokai) No. 10-87493), isoxazole derivatives (Japanese Unexamined PatentPublication (Kokai) No. 11-1479), imidazolidine derivatives(WO96-04248), hydantoin derivatives (Japanese Unexamined PatentPublication (Kokai) No. 9-31061), quinazoline derivatives (WO97-11941),etc., but as a typical examples of a preferable chymase inhibitor, acompound of the following formula (I) and its pharmaceuticallyacceptable salts may be mentioned.

wherein, the ring A represents an aryl group;

R¹ represents a hydroxyl group, an amino group, a C₁ to C₄ loweralkylamino group which may be substituted with a carboxylic acid group,a C₇ to C₁₀ lower aralkylamino group which may be substituted with acarboxylic acid group, an amino group acylated with a C₁ to C₄ loweraliphatic acid which may be substituted with a carboxylic acid group, anamino group acylated with an aromatic ring carboxylic acid which may besubstituted with a carboxylic acid group, an amino group acylated with aheteroaromatic ring carboxylic acid which may be substituted with acarboxylic acid group, an amino group sulfonylated with a C₁ to C₄ loweralkanesulfonic acid which may be substituted with a carboxylic acidgroup, an amino group sulfonylated with an aromatic ring sulfonic acidwhich may be substituted with a carboxylic acid group, an amino groupsulfonylated with a heteroaromatic ring sulfonic acid which may besubstituted with a carboxylic acid group, a C₁ to C₄ lower alkyl groupsubstituted with a carboxylic acid group, or a C₂ to C₄ lower alkylenegroup which may be substituted with a carboxylic acid group;

R² and R³ may be the same or different and represent a hydrogen atom, anunsubstituted or substituted C₁ to C₄ lower alkyl group, a halogen atom,a hydroxyl group, a C₁ to C₄ lower alkoxyl group, an amino group, anunsubstituted or substituted C₁ to C₄ lower alkylamino group, anunsubstituted or substituted C₇ to C₁₀ aralkylamino group, an aminogroup acylated with a C₁ to C₄ lower aliphatic acid which may besubstituted with a carboxylic acid group, an amino group acylated withan aromatic ring carboxylic acid which may be substituted with acarboxylic acid group, an amino group acylated with a heteroaromaticring carboxylic acid which may be substituted with a carboxylic acidgroup, an amino group sulfonylated with a C₁ to C₄ lower alkanesulfonicacid which may be substituted with a carboxylic acid group, an aminogroup sulfonylated with an aromatic ring sulfonic acid which may besubstituted with a carboxylic acid group, an amino group sulfonylatedwith a heteroaromatic ring sulfonic acid which may be substituted with acarboxylic acid group, or a carboxylic acid group or when the ring A isa benzene ring, R¹ and R² may form, together with the substitutingbenzene ring, a fused heterocyclic ring which may be substituted with acarboxylic acid and in which the carbon atom in the ring may form acarbonyl group and R³ is the same as defined above; and

X represents a hydrogen atom, a C₁ to C₄ lower alkyl group, a C₁ to C₄lower alkoxy group, a halogen atom, a hydroxyl group, an amino group, ora nitro group.

In the general formula (I), preferable examples of the aryl grouprepresented by the ring A are a benzene ring and a naphthalene ring.

Preferable examples of the C₁ to C₄ lower alkylamino group which may besubstituted with the carboxylic acid group and the C₇ to C₁₂ loweraralkylamino group which may be substituted with a carboxylic acid grouprepresented by R¹ are a methylamino group, an ethylamino group, apropylamino group, a butylamino group, a carboxymethylamino group, acarboxyethylamino group, a carboxypropylamino group, a carboxybutylaminogroup, a benzylamino group, a phenetylamino group, a phenylpropylaminogroup, a phenylbutylamino group, a carboxybenzylamino group, acarboxyphenetylamino group, a carboxyphenylpropylamino group, acarboxyphenylbutylamino group, etc.

Preferable examples of the amino group acylated with a C₁ to C₄ loweraliphatic acid which may be substituted with a carboxylic acid group,the amino group acylated with an aromatic ring carboxylic acid which maybe substituted with a carboxylic acid group, and the amino groupacylated with a heteroaromatic ring carboxylic acid which may besubstituted with a carboxylic acid group represented by R¹ are aformylamino group, an acetylamino group, a propionylamino group, abutyrylamino group, a benzoylamino group, a naphthoylamino group, apyridinecarbonylamino group, a pyrrolecarbonylamino group, acarboxyacetylamino group, a carboxypropionylamino group, acarboxybutyrylamino group, a carboxybenzoylamino group, acarboxynaphthoylamino group, a carboxypyridinecarbonylamino group, acarboxypyrrolecarbonylamino group, etc.

Preferable examples of the amino group sulfonylated with a C₁ to C₄lower alkanesulfonic acid which may be substituted with a carboxylicacid group, the amino group sulfonylated with an aromatic ring sulfonicacid which may be substituted with a carboxylic acid group, and theamino group sulfonylated with a heteroaromatic ring sulfonic acid whichmay be substituted with a carboxylic acid group represented by R¹ are amethanesulfonylamino group, an ethanesulfonylamino group, apropanesulfonylamino group, a butanesulfonylamino group, abenzenesulfonylamino group, a naphthalenesulfonylamino group, apyridinesulfonylamino group, a pyrrolesulfonylamino group, acarboxymethanesulfonylamino group, a carboxyethanesulfonylamino group, acarboxypropanesulfonylamino group, a carboxybutane-sulfonylamino group,a carboxybenzenesulfonylamino group, a carboxynaphthalenesulfonylaminogroup, a carboxypyridinesulfonylamino group, acarboxypyrrolesulfonylamino group, etc.

Preferable examples of the C₁ to C₄ lower alkyl group substituted with acarboxylic acid group represented by R¹ are an acetic acid group, apropionic acid group, a butyric acid group, a valeric acid group, etc.

Preferable examples of the C₂ to C₄ lower alkylene group substitutedwith a carboxylic acid group represented by R¹ are an acrylic acidgroup, a crotonic acid group, etc.

Preferable examples of the unsubstituted or substituted C₁ to C₄ loweralkyl group represented by R² or R³ are a straight-chain alkyl groupsuch as a methyl group, an ethyl group, a n-propyl group, and a n-butylgroup and a branched alkyl group such as an isopropyl group, a sec-butylgroup, and a t-butyl group.

Preferable examples of the substituent group of the C₁ to C₄ lower alkylgroup are a carboxylic acid group, a halogen atom such as a fluorineatom and a chlorine atom, a C₁ to C₄ lower alkoxy group, an amino group,a methylamino group, a dimethylamino group, a carboxymethylamino group,a carboxyethylamino group, etc.

Preferable examples of the halogen atom represented by R² or R³ are afluorine atom, a chlorine atom, a bromine atom and an iodine atom.

Preferable examples of the C₁ to C₄ lower alkoxyl group represented byR² or R³ are a straight-chain alkyloxy group such as a methoxy group, anethoxy group, a n-propyloxy group, and a n-butoxy group and a branchedalkyloxy group such as an isopropyloxy group, a sec-butoxy group, and at-butoxy group.

Preferable examples of the unsubstituted or substituted C₁ to C₄ loweralkylamino group represented by R² or R³ are a methylamino group, anethylamino group, a propylamino group, a butylamino group, etc.

Preferable examples of the substituent group of the C₁ to C₄ loweralkylamino group are a carboxylic acid group, a halogen atom such as afluorine atom and a chlorine atom, a C₁ to C₄ lower alkoxyl group, etc.

Preferable examples of the unsubstituted or substituted C₇ to C₁₂ loweraralkylamino group represented by R² or R³ are a benzylamino group, aphenetylamino group, a phenylpropylamino group, a phenylbutylaminogroup, etc.

Preferable examples of the substituent group of the aralkylamino groupare a carboxylic acid group, a halogen atom such as a fluorine atom anda chlorine atom, a C₁ to C₄ lower alkoxyl group, etc.

Preferable examples of the amino group acylated with a C₁ to C₄ loweraliphatic acid which may be substituted with a carboxylic acid group,the amino group acylated with an aromatic ring carboxylic acid which maybe substituted with a carboxylic acid group, and the amino groupacylated with a heteroaromatic ring carboxylic acid which may besubstituted with a carboxylic acid group represented by R² or R³ are aformylamino group, an acetylamino group, a propionylamino group, abutyrylamino group, a benzoylamino group, a naphthoylamino group, apyridinecarbonylamino group, a pyrrolecarbonylamino group, acarboxyacetylamino group, a carboxypropionylamino group, acarboxybutyrylamino group, a carboxybenzoylamino group, acarboxynaphthoylamino group, a carboxypyridinecarbonylamino group, acarboxypyrrolecarbonylamino group, etc.

Preferable examples of the amino group sulfonylated with a C₁ to C₄lower alkanesulfonic acid which may be substituted with a carboxylicacid group, the amino group sulfonylated with an aromatic ring sulfonicacid which may be substituted with a carboxylic acid group, and theamino group sulfonylated with a heteroaromatic ring sulfonic acid whichmay be substituted with a carboxylic acid group represented by R² or R³are a methanesulfonylamino group, an ethanesulfonylamino group, apropanesulfonylamino group, a benzenesulfbnylamino group, anaphthalenesulfonylamino group, a pyridinesulfonylamino group, apyrrolesulfonylamino group, a carboxymethanesulfonylamino group, acarboxyethanesulfonylamino group, a carboxypropanesulfonylamino group, acarboxybenzenesulfonylamino group, a carboxynaphthalenesulfonylaminogroup, a carboxypyridine-sulfonylamino group, acarboxypyrrolesulfonylamino group, etc.

Preferable examples of the fused heterocyclic ring which may besubstituted with a carboxylic acid and in which the carbon atom in thering may form a carbonyl group which R¹ and R² form together with thesubstituting benzene ring when the ring A is a benzene ring, are atetrahydroquinoline ring and a benzoxazine ring, for example, atetrahydroquinoline, a benzoxazine, a quinoxaline, a benzodioxane, acarboxytetrahydroquinoline, a carboxybenzoxazine, a carboxyquinoxaline,a carboxybenzodioxane, etc.

Preferable examples of the C₁ to C₄ lower alkyl group represented by Xare a straight-chain alkyl group such as a methyl group, an ethyl group,a n-propyl group, and a n-butyl group and a branched alkyl group such asan isopropyl group, a sec-butyl group, and a t-butyl group.

Preferable examples of the C₁ to C₄ lower alkoxyl group represented by Xare a straight-chain alkyloxy group such as a methoxy group, an ethoxygroup, a n-propyloxy group, and a n-butoxy group and a branched alkyloxygroup such as an isopropyloxy group, a sec-butoxy group, and a t-butoxygroup.

Preferable examples of the halogen atom represented by X, are a fluorineatom, a chlorine atom, a bromine atom and an iodine atom.

Further, examples of a pharmaceutically acceptable salts are an acidsalt such as a hydrochloric acid salt, a methanesulfonic acid salt, anda trifluoroacetic acid salt and an alkali metal salt such as a sodiumsalt and a potassium salt.

The other typical examples of the preferable chymase inhibitor are aquinazoline derivative having the formula (II) and its pharmaceuticallyacceptable salts:

wherein, the ring B represents a benzene ring, a pyridine ring, apyrrole ring, or a pyrazole ring, m represents 0, 1, or 2,

Y represents a hydroxy group, a nitro group, a halogen atom, a C₁ to C₄lower alkyl group which may be substituted with a halogen atom, a C₁ toC₄ lower alkoxy group which may be substituted with a halogen atom, or aC₇ to C₁₂ aralkyloxy group, or Y represents a group forming anaphthalene ring or a quinoline ring together with the benzene ringwhich is shown as substituted with said Y,

R⁵ and R⁶ are the same or different and represent a hydrogen atom, ahalogen atom, a C₁ to C₄ lower alkyl group which may be substituted witha halogen atom, a nitro group, a cyano group, a pyrazolyl group, atetrazolyl group, a carboxyl group which may be esterified with a C₁ toC₄ lower alkyl group or an allyl group, or a C₁ to C₄ lower alkoxy groupwhich may be substituted with one or more substituent groups selectedfrom the group consisting of a halogen atom, a morpholino group, aphenylpiperazinyl group, and a carboxyl group which may be esterifiedwith a C₁ to C₄ lower alkyl group or an allyl group, or, when the ring Brepresents a benzene ring, R⁵ and R⁶ represent a group forming anaphthalene ring or a quinoline ring together with the benzene ringwhich is shown as substituted with said R⁵ and R⁶, and

Z represents a hydrogen atom, a C₁ to C₄ lower alkyl group which may besubstituted with a halogen atom, a C₂ to C₅ alkenyl group, anunsubstituted or substituted aralkyl group, an unsubstituted orsubstituted aromatic heterocyclic alkyl group, a carboxylmethyl groupwhich may be esterified with a C₁ to C₄ lower alkyl group or an allylgroup, a carbonylmethyl group which is amidated with a primary orsecondary or cyclic amine, an unsubstituted or substitutedarylcarbonylmethyl group, or an unsubstituted or substitutedaralkyloxymethyl group.

In the general formula (II), the preferable examples of the halogen atomfor Y are fluorine, chlorine, bromine, or iodine. The examples of thelower alkyl group of the C₁ to C₄ lower alkyl group for Y, which issubstituted with a halogen atom, are straight chain alkyl groups such asa methyl group, ethyl group, n-propyl group and n-butyl group, andbranched alkyl groups such as an isopropyl group, sec-butyl group andt-butyl group, and, while the examples of the halogen atom of the C₁ toC₄ lower alkyl group for Y which is substituted with a halogen atom, arefluorine, chlorine, bromine and iodine. The examples of the lower alkoxygroup of the C₁ to C₄ lower alkoxy group for Y, which is substitutedwith a halogen atom, are straight chain alkoxy groups such as a methoxygroup, ethoxy group, n-propoxy group and n-butoxy group, and branchedalkoxy groups such as an isopropoxy group, sec-butoxy group and t-butoxygroup, while the examples of the halogen atom of the C₁ to C₄ loweralkoxy group for Y, which is substituted with a halogen atom, arefluorine, chlorine, bromine, and iodine. The examples of the C₇ to C₁₂aralkyloxy group for Y are a benzyloxy group, phenethyloxy group,phenylpropoxy group and naphthylethyloxy group, etc, preferably thebenzyloxy group.

The preferable examples of the halogen atom for R⁵ or R⁶ are fluorine,chlorine, bromine, or iodine. The examples of the lower alkyl group ofthe C₁ to C₄ lower alkyl group for R⁵ or R⁶, which is substituted with ahalogen atom, are straight chain alkyl groups such as a methyl group,ethyl group, n-propyl group and n-butyl group, and branched alkyl groupssuch as an isopropyl group, sec-butyl group and t-butyl group, while theexamples of the halogen atom of the C₁ to C₄ lower alkyl group for R⁵ orR⁶, which is substituted with a halogen atom, are fluorine, chlorine,bromine, or iodine. The preferable examples of the C₁ to C₄ lower alkylgroup of the carboxyl group for R⁵ or R⁶, which may be esterified withthe C₁ to C₄ lower alkyl group or an allyl group, are straight chainalkyl groups such as a methyl group, ethyl group, n-propyl group andn-butyl group, and branched alkyl groups such as an isopropyl group,sec-butyl group and t-butyl group. The examples of the alkoxy group ofthe C₁ to C₄ lower alkoxy group for R⁵ or R⁶, which is substituted withone or more substituent groups selected from the group consisting of ahalogen atom, a morpholino group, a phenylpiperazinyl group, and acarboxyl group which may be esterified with a C₁ to C₄ lower alkyl groupor an allyl group, are straight chain alkoxy groups such as a methoxygroup, ethoxy group, n-propoxy group and n-butoxy group, and branchedalkoxy groups such as an isopropoxy group, sec-butoxy group and t-butoxygroup. The examples of the halogen atom shown as the above substituentgroup, are fluorine, chlorine, bromine, or iodine and the preferableexamples of the C₁ to C₄ lower alkyl group of the carboxyl group whichis esterified with a C₁ to C₄ lower alkyl group or an allyl group shownas the above substituent group, are a methyl group, ethyl group,n-propyl group, n-butyl group, and other straight chain alkyl groups andan isopropyl group, sec-butyl group, t-butyl group, and other branchedalkyl groups.

The examples of the lower alkyl group of the C₁ to C₄ lower alkyl groupshown as Z which may be substituted with halogen, are straight chainalkyl groups such as a methyl group, ethyl group, n-propyl group andn-butyl group, and branched alkyl groups such as an isopropyl group,sec-butyl group and t-butyl group, while the examples of the halogenatom of the C₁ to C₄ lower alkyl group which may be substituted with thehalogen atom are fluorine, chlorine, bromine, or iodine. The examples ofthe C₂ to C₅ alkenyl group for Z are an allyl group, propenyl group,isopropenyl group, butenyl group, etc.

The examples of the aralkyl group of an unsubstituted or substitutedaralkyl group shown as Z are a C₇ to C₁₂ aralkyl group, preferably abenzyl group, phenethyl group, phenylpropyl group, or naphthylethylgroup. The preferable examples of the substituent group of anunsubstituted or substituted aralkyl group are a carboxyl group whichmay be esterified with a C₁ to C₄ lower alkyl group or an allyl group, acyano group, a nitro group, a carbonyl group amidized with primaryamine, an amine group which may be amidized with a carboxylic acid or anamino acid, and a guanidino group which may be substituted with a loweralkoxycarbonyl group. The examples of the lower alkyl group of thecarboxyl group, which may be esterified with a C₁ to C₄ lower alkylgroup or an allyl group are straight alkyl groups such as a methylgroup, ethyl group, n-propyl group and n-butyl group, and branched alkylgroups such as an isopropyl group, sec-butyl group and t-butyl group.The examples of the primary amine of the carbonyl group amidized withprimary amine are a chain C₁ to C₄ lower alkylamine or those which maybe substituted with carboxyl group, such as, preferably, methylamine,ethylamine, isopropylamine and carboxylmethylamine; amines havingmonocyclic or polycyclic aromatic hydrocarbon group such as aniline andnaphthylamine; amines having aromatic heterocyclic group such asaminopyridine, amihopyrrole, and other. The examples of the carboxylicacid of the amine group which may be amidized with a carboxylic acid oran amino acid are preferably C₂ to C₅ aliphatic monocarboxylic acids oraliphatic dicarboxylic acids such as pivalic acid and succinic acid,while the examples of the amino acid are amino acids, of which carboxylgroup may be esterified or of which amine group may be amidized, such asL-aspartic acid, α-O-t-butyl-N-t-butoxycarbonyl-L-aspartic acid andother. The examples of the guanidino group which may be substituted witha lower alkoxycarbonyl group are preferably a guadinino group which maybe substituted with a C₂ to C₁ lower alkoxycarbonyl group such as aguanidino group and 2,3-bis-t-butoxycarbonylguanidino group.

The examples of the aromatic heterocyclic alkyl group of anunsubstituted or substituted aromatic heterocyclic alkyl group shown asZ are thienylalkyl groups such as a 2-thenyl group and a 2-thienylethylgroup, furylalkyl groups such as a 2-furfuryl group and a 2-furylethylgroup, pyridylalkyl groups such as a 2-pyridylmethyl group,3-pyridylmethyl group, 4-pyridylmethyl group and 4-pyridylethyl group,pyrimidinylalkyl groups such as a 5-pyrimidinylmethyl group,pyrazinylalkyl groups such as a 2-pyrazinylmethyl group,pyridazinylalkyl groups such as a 3-pyridazinylmethyl grouptetrazolylalkyl groups such as a 5-tetrazolylmethyl group,isothiazolylalkyl groups such as a 4-isothiazolylmethyl group and a5-isothiazolylmethyl group, thiazolylalkyl groups such as a5-thiazolylmethyl group, oxazolylalkyl groups such as a 5-oxazolylmethylgroup, and isoxazolylalkyl groups such as a 4-isooxazolylmethyl groupand 5-isoxazolylmethyl group. The preferable examples of the substituentgroup of an unsubstituted or substituted heterocyclic alkyl group, areC₁ to C₄ lower alkyl groups such as a methyl group and ethyl group, andC₁ to C₄ carboxyl lower alkyl groups such as a carboxylmethyl group andcarboxylethyl group.

The examples of the lower alkyl group of the carboxymethyl group whichmay be esterified with a C₁ to C₄ lower alkyl group or an allyl groupshown as Z are straight chain alkyl groups such as a methyl group, ethylgroup, n-propyl group and n-butyl group, and branched alkyl groups suchas an isopropyl group, sec-butyl group and t-butyl group.

The examples of the primary amine of the carbonylmethyl group which maybe amidized with primary or secondary or cyclic amine shown as Z arechain C₁ to C₄ lower alkylamines or those which may be substituted witha carboxyl group such as preferably methylamine, ethylamine,isopropylamine and carboxylmethylamine, and amines having monocyclicsaturated hydrocarbon group such as a cyclohexylamine, and amines havingmonocyclic or polycyclic aromatic hydrocarbon group such as aniline, abenzylamine and a naphthylamine, and amines having aromatic heterocyclicgroup such as an aminopyridine, an aminomethylpyridine, an aminopyrrole,an aminopyrimidine, an aminoindole and aminoquinoline, wherein theamines having aromatic hydrocarbon group or aromatic heterocyclic groupmay have on its ring one or more substituent such as

1) hydroxy group,

2) —OPO (OH)₂,

3) amino group,

4) oxo group,

5) halogen atom,

6) carboxyl group, which may be esterified with C₁ to C₄ lower alkylgroup such as a methyl group, an ethyl group, an isopropyl group and at-butyl group, or an allyl group,

7) straight chain or branched C₁ to C₄ lower alkoxy group such as amethoxy group, an ethoxy group, a n-propoxy group and t-butoxy group,which may be substituted with a carboxyl group, which may be esterifiedwith a C₁ to C₄ lower alkyl group such as a methyl group, an ethylgroup, an isopropyl group and a t-butyl group, or an allyl group,

8) straight chain or branched C₁ to C₄ lower alkyl groups such as amethyl group, an ethyl group, a n-propyl group, a n-butyl group, anisopropyl group, a sec-butyl group, and t-butyl group, which may besubstituted.

Further, the preferable examples of the substituent of the C₁ to C₄lower alkyl group, which may be substituted, of the above 8),

a) a carboxyl group, which may be esterified with C₁ to C₄ lower alkylgroup such as a methyl group, an ethyl group, an isopropyl group and at-butyl group, or an allyl group,

b) piperadinyl group, which may be N-substituted with carboxy groupwhich is esterified with C₁ to C₄ lower alkyl group such as a methylgroup, an ethyl group, an isopropyl group and a t-butyl group, or anallyl group,

c) morpholino group, and

d) amino group which may be amidized with carboxylic acid or amino acid

The examples of the carboxylic acid of amino group of the above d),which may be amidized with carboxylic acid or amino acid, are preferablyC₂ to C₅ aliphatic mono- or di-carboxylic acids such as pivalic acid andsuccinic acid, while the examples of the amino acid are amino acids ofwhich carboxyl group may be esterified or of which amino group may beamidized, such as a L-aspartic acid, anα-O-t-butyl-N-t-butoxycarbonyl-L-aspartic acid, and aβ-O-t-butyl-N-t-butoxycarbonyl-L-aspartic acid. Further, the aminehaving aromatic heterocyclic group may have the nitrogen atom on itsring, which may be substituted with C₁ to C₄ lower alkyl group such as amethyl group, and an ethyl group, or carboxy lower alkyl group, whichmay be esterified, such as a carboxylmethyl group and a t-butoxycarbonylmethyl group.

The examples of the secondary amine of the carbonylmethyl group shown asZ, which is amidized with primary or secondary or cyclic amine aredi-lower alkylamines such as a dimethylamine and diethylamine. Theexamples of the cyclic amine of the carbonylmethyl group shown as Z,which is amidized with primary or secondary or cyclic amine arepyrrolidine and piperidine.

The examples of the arylcarbonylmethyl group of the unsubstituted orsubstituted arylcarbonylmethyl group shown as Z are aphenylcarbonylmethyl group and a naphthylcarbonylmethyl group, while thepreferable examples of the substituent group are a hydroxy group, anitro group, halogen atoms such as fluorine, chlorine, bromine andiodine, straight or branched C₁ to C₄ lower alkyl groups, which may besubstituted with halogen atom such as methyl group, ethyl group,n-propyl group, n-butyl group, isopropyl group, sec-butyl group andt-butyl group, straight or branched C₁ to C₄ lower alkoxy groups, whichmay be substituted with halogen atom, such as methoxy group, ethoxygroup, n-propoxy group, n-butoxy group, isopropoxy group, sec-butoxygroup and t-butoxy group.

The examples of the aralkyloxymethyl group of the unsubstituted orsubstituted aralkyloxymethyl group shown as Z, are preferably C₈ to C₁₃aralkyloxymethyl groups such as a benzyloxy methyl group,phenethyloxymethyl group and naphthylethyloxymethyl group, while thepreferable examples of the substituent group are a hydroxy group, anitro group, halogen atoms such as fluorine, chlorine, bromine andiodine, straight or branched C₁ to C₄ lower alkyl groups which may besubstituted with halogen atom, such as methyl group, ethyl group,n-propyl group, n-butyl group, isopropyl group, sec-butyl group andt-butyl group, straight or branched C₁ to C₄ lower alkoxy groups, whichmay be substituted with halogen atom, such as methoxy group, ethoxygroup, n-propoxy group, n-butoxy group, isopropoxy group, sec-butoxygroup and t-butoxy group.

Further, the examples of a pharmaceutically acceptable salt are acidsalts such as a chlorate and nitrate and alkali metal salts such as asodium salt, potassium salt.

The quinazoline derivative having the formula (I) according to thepresent invention may, for example, be synthesized by the followingSynthesis Method (A) or (B).

Synthesis Method (A)

A compound having the formula (I-1):

wherein the ring A is the same as defined above and R¹, R²′ and R³′represent R¹, R² and R³, which may be protected with a protecting group,respectively, and R¹, R² and R³ represent the same as defined above

is reacted with an anthranilic acid derivative having the formula (I-2):

wherein X′ represents X, which may be protected with a protecting group,and X represents the same as defined above

using the method described, for example, in JP-A-6-199839 to obtain asulfonylurea derivative having the formula (I-3):

wherein the ring A, R^(1′), R^(2′), R^(3′) and X′ represent the same asdefined above,

then, a condensing agent for example, 1,1′-carbonyldiimidazole(hereinafter referred to as CDI) is used to obtain the quinazoline ring,and if necessary, the protecting groups of R¹, R², R³ and X aredeprotected.

In this reaction, when R¹, R² or R³ represents a group containing ahydroxyl group, an amino group, or a carboxylic acid group, R¹, R² or R³may be optionally protected by a protecting group such as abenzyloxycarbonyl group, a t-butoxycarbonyl group, a benzyl group, anallyl group, a t-butyl group, etc. When X represents a hydroxyl group oran amino group, X may be optionally protected with a protecting groupsuch as a benzyloxycarbonyl group, a t-butoxycarbonyl group, a benzylgroup, an allyl group, a t-butyl group, etc.

The compound having the formula (I-1) used in this reaction includes acommercially available or known compound or a compound which can besynthesized by a known method may be used. For example, using thesynthesis method described in the specification of European Patent No.0269141, it is possible to use a compound which can be synthesized fromthe corresponding sulfonamide derivative using chlorosulfonylisocyanate. For example, it is possible to use3-allyloxycarbonyl-methylbenzenesulfonyl isocyanate,4-allyloxycarbonyl-methylbenzenesulfonyl isocyanate,4-allyloxybenzenesulfonyl isocyanate, etc.

As the anthranilic acid derivative having the formula (I-2) used forthis reaction, a commercially available or known compound or a compoundwhich can be synthesized by a known method may be used. For example,anthranilic acid, 4-chloroanthranilic acid, 4-methoxyanthranilic acid,5-chloroanthranilic acid, 4-hydroxyanthranilic acid, etc. may be used.

The reaction to obtain the quinazoline ring from the sulfonylureaderivative having the formula (I-3) may be carried out using anaprotonic solvent such as, for example, an ether solvent such astetrahydrofuran and dioxane, a halogen-containing solvent such asmethylene chloride, or dimethylformamide etc. at a temperature of −50°C. to 50° C., preferably −20° C. to room temperature. Further, for thecyclization reaction, it is possible to use an ordinary condensing agentwhich includes, for example, CDI, dicyclohexylcarbodiimide, and similarcarbodiimide compounds, mixed anhydrides, etc. The deprotecting reactioncan be carried out by an ordinary method using hydrolysis with an acidor alkali, reduction or oxidation etc.

Synthesis Method (B)

A compound having the formula (I-4):

wherein the ring A, R^(1′), R^(2′) and R^(3′) represent the same asdefined above

is condensed with an anthranilic acid derivative having the formula(I-5):

wherein X′ represents the same as defined above, Ph represents a phenylgroup, and R⁴ represents a protecting group of the carboxyl group, whichis specifically a group capable of being released by hydrolysis orhydrogenolysis, such as, for example, a methyl group, an ethyl group, ora benzyl group

using, for example, 1,8-diazabicyclo[5,4,0]-7-undecene (hereinafterreferred to as DBU) to form a sulfonylurea derivative having the formula(I-6):

wherein the ring A, R^(1′), R^(2′), R^(3′), R⁴ and X′ are the same asdefined above,

which is then hydrolyzed with an alkali or hydrogenolyzed to derive acorresponding carboxylic acid represented by the formula (I-3), then thequinazoline ring is obtained and optionally the protecting groups of R¹,R², R³ and x are deprotected, in the same way as in Synthesis Method(A). In this reaction, when R¹, R² or R³ represents a group containing ahydroxyl group, an amino group, or a carboxylic acid group, R¹, R² or R³may be optionally protected by a protecting group such as abenzyloxycarbonyl group, a t-butoxycarbonyl group, a benzyl group, anallyl group, a t-butyl group, etc. When X represents a hydroxyl group oran amino group, X may be optionally protected with a protecting groupsuch as a benzyloxycarbonyl group, a t-butoxycarbonyl group, a benzylgroup, an allyl group, a t-butyl group, etc.

As the compound having the formula (I-4) used in the reaction, acommercially available or known compound or a compound which can besynthesized by a known method may be used. For example,3-hydroxybenzenesulfonamide, 2-aminobenzenesulfonamide,3-aminobenzenesulfonamide, 4-aminobenzenesulfonamide,(±)-2-(4-aminosulfonylphenyl)butyric acid,3-benzyloxycarbonylamino-4-chlorobenzenesulfonamide,4-benzyloxycarbonylamino-3-chlorobenzenesulfonamide,4-amino-3,5-dichlorobenzenesulfonamide,3-benzyloxycarbonylamino-4-methylbenzenesulfonamide,4-t-butoxycarbonyl-3-hydroxybenzenesulfonamide,3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonamide,4-t-butoxycarbonyl-3-hydroxybenzenesulfonamide,3-t-butoxycarbonyl-4-hydroxybenzenesulfonamide,3-acetamide-4-methoxybenzenesulfonamide,3-(3-aminosulfonyl)phenylacrylic acid t-butylester,3-amino-4-methoxybenzenesulfonamide,4-methoxy-3-methylsulfonylaminobenzenesulfonamide,3-carboxy-4-hydroxy-2-naphthalenesulfonamide,4-benzyloxycarbonylamino-3-t-butoxycarbonylbenzenesulfonamide,(±)-3-t-butoxycarbonyl-2-oxo-1H,3H-quinoline-7-sulfonamide,(±)-2-t-butoxycarbonyl-3-oxo-1,4-benzoxazine-6-sulfonamide, etc. may beused.

As the anthranilic acid derivative having the formula (I-5) used in thisreaction, a commercially available or known compound or a compound whichcan be synthesized by a known method may be used. For example, methyl4-chloro-2-N-phenoxycarbonylanthranilate, ethyl4-chloro-2-N-phenoxycarbonylanthranilate, benzyl4-chloro-2-N-phenoxycarbonylanthranilate, methyl5-chloro-2-N-phenoxycarbonylanthranilate, ethyl5-chloro-2-N-phenoxycarbonylanthranilate, benzyl5-chloro-2-N-phenoxycarbonylanthranilate, methyl4-methoxy-2-N-phenoxycarbonylanthranilate, ethyl4-methoxy-2-N-phenoxycarbonylanthranilate, benzyl4-methoxy-2-N-phenoxycarbonylanthranilate, methyl4-hydroxy-2-N-phenoxycarbonylanthranilate, ethyl4-hydroxy-2-N-phenoxycarbonylanthranilate, benzyl4-hydroxy-2-N-phenoxycarbonylanthranilate, etc. may be used.

The reaction for obtaining the compound having the formula (I-4) and theanthranilic acid derivative having the formula (I-5) condense to obtaina sulfonylurea derivative having the formula (I-6), may be carried outusing an aprotic solvent, for example, an ether solvent such astetrahydrofuran or dioxane, a halogen-containing solvent such asmethylene chloride, or dimethylformamide etc. at a temperature of −50°C. to 50° C., preferably −20° C. to room temperature. Further, as theusable for the condensation reaction, an organic strong base such asDBU, inorganic bases such as potassium carbonate, sodium carbonate,potassium hydroxide, and sodium hydroxide, or metal bases such as sodiumhydride may be used.

In the reaction for alkali hydrolysis or hydrogenolysis of thesulfonylurea derivative having the formula (I-6) thus obtained to obtainthe sulfonylurea derivative having the formula (I-3), ordinaryhydrolysis conditions or hydrogenolysis conditions for esters may beused.

Note that the above reaction may be carried out while protecting thefunctional groups not involved in the reaction. According to the type ofthe protecting group, the protection is removed by chemical reduction orother ordinary protection-removing reactions. For example, when theprotecting group is a t-butyl group or t-butoxycarbonyl group,trifluoroacetic acid may be used, while when it is an allyl group,palladium catalysts such as tetrakis(triphenylphosphine)palladium (0)may be used.

The compound having the formula (I), wherein R¹ represents an aminogroup acylated with a C₁ to C₄ lower aliphatic acid which may besubstituted with a carboxylic acid, an amino group acylated with anaromatic ring carboxylic acid which may be substituted with a carboxylicacid and an amino group acylated with an heteroaromatic ring carboxylicacid which may be substituted with a carboxylic acid, can be obtainedfrom the compound having the formula (I), wherein R¹ represents an aminogroup, by acylating the same with carboxylic acid, carboxylic acidchloride, carboxylic acid anhydride using an ordinary method.

The compound having the formula (I), wherein R¹ represents an aminogroup sulfonylated with a C₁ to C₄ lower alkane sulfonic acid which maybe substituted with a carboxylic acid, an amino group sulfonylated withan aromatic ring sulfonic acid which may be substituted with acarboxylic acid and an amino group sulfonylated with an heteroaromaticring sulfonic acid which may be substituted with a carboxylic acid, canbe obtained from the compound having the formula (I), wherein R¹represents an amino group, by sulfonylating the same with sulfonic acidor sulfonic acid chloride using an ordinary method.

A compound of formula (II) of the present invention may be obtained by asimilar method to the above and further is as described in more detailin International Publication WO97/11941.

A compound of the formula (I) or (II) obtained may be purified by aconventional method such as recrystallization or column chromatography.

Further, as necessary, the compound of the formula (I) or (II) obtainedby the above process may be converted into a salt by causing it to reactwith various types of acids or bases. As the acid able to be used toconvert the compound of formula (I) or (II) to a salt, an inorganic acidsuch as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid,or phosphoric acid and an organic acid such as methanesulfonic acid,benzenesulfonic acid, p-toluene sulfonic acid, trifluoroacetic acid,citric acid, lactic acid, maleic acid, fumaric acid, tartaric acid,acetic acid, adipic acid, palmitic acid, and tannic acid may bementioned.

As the base able to be used for converting the compound of formula (I)or (II) into a salt, sodium hydroxide, lithium hydroxide, and potassiumhydroxide may be mentioned.

The compounds having the formula (I) or (II) include ones containingasymmetric centers. It is possible to isolate a single optically activesubstance from the racemic mixture by one or more methods. For example,

(1) the method of using an optically active column

(2) the method of conversion to a salt by an optically active acid orbase and then recrystallization

(3) the method combining the above (1) and (2) may be used.

These compounds may be evaluated for action in preventing or alleviatingconditions of dermatitis exhibiting biphasic skin reaction anddermatitis induced, by repeated exposure to an antigen by the laterexplained methods of Examples 3, 8, 10, 11, 12, and 18.

When using a compound according to the present invention as a medicamentfor the prevention or treatment of dermatitis exhibiting biphasic skinreaction, a medicament for the alleviation of the late-phase reaction ofdermatitis exhibiting biphasic skin reaction, or a medicament forprevention or treatment of dermatitis induced by repeated exposure to anantigen, for example it is possible to use one type or a mixture of twoor more types of the compound of the present invention to make apreparation of a form suitable for the method of administrationaccording to an ordinary method. For example, examples of preparationforms for oral administration include capsules, tablets, granules, finegranules, syrups, dry syrups, and other preparations, while examples ofpreparation forms for non-oral administration include injections andalso suppositories such as rectal suppositories and vaginalsuppositories, transnasal preparations such as sprays and ointments, andtransdermal preparations such as tapes for transdermal absorption.

The clinical dose of the compound according to the present inventionvaries according to the symptoms, severity, age, presence ofcomplications, etc. and also varies according to the form ofpreparation. In the case of oral administration, however, it may bedosed usually, in terms of effective ingredients, as 1 to 1000 mg peradult per day. In the case of non-oral administration, it is sufficientto administer 1/10 to ½ the amount of the case of oral administration.These dosages can be suitably adjusted according to the age, symptoms,etc. of the patient.

In the present invention, the chymase inhibitor can be administeredalone as it is without being mixed with another effective ingredient,but considering the disease in question, the symptoms, complications,etc., it may also administered as a medicinal preparation containingother effective ingredients. Further, it may also be combined with theseother effective ingredients. The amounts of the other effectiveingredients used are not particularly limited, but are determinedconsidering the minimum amounts for expression of their effects alone,the occurrence of side effects, etc.

In treatment, the form of preparation and the method of combinedtreatment including preparations containing the chymase inhibitor aloneas an effective ingredient and preparations also containing othereffective ingredients are suitably selected by a physician in accordancewith the age of the patient, the symptoms, etc.

The toxicity of the compound according to the present invention is low.The acute toxicity value LD₅₀ at 24 hours after oral administration to5-week old male mice was 1 g/kg or more. This value is more than 50times the anticipated clinical dosage. The compound is therefore judgedto be high in safety.

EXAMPLES

The present invention will now be further explained by, but is by nomeans limited to, the following Examples, but the scope of the inventionis not limited to these Examples needless to say.

In Example 2 and Example 3, a mouse model of dermatitis exhibitingbiphasic skin reaction was used to show the usefulness of a chymaseinhibitor by showing the effect of suppression of the chymase inhibitor.Further, in Example 4 to Example 6, as further proof supporting theinvolvement of chymase in dermatitis exhibiting biphasic skin reaction,the fact that dermatitis exhibiting biphasic skin reaction is induced byintradermal inoculation of human chymase into the ear of mice ispresented. In Example 7 and Example 8, the results of analysis of themechanism of action of chymase in such biphasic skin reaction are shown.

On the other hand, in Example 9 to Example 12, the usefulness of achymase inhibitor is demonstrated by using dermatitis induced byrepeated application of hapten as a model for dermatitis induced byrepeated exposure to an antigen and analyzing the model and showing theeffect of a chymase inhibitor in this model. Further, since it isconceivable that mast cells undergo repeated degranulation reactions dueto repeated exposure to allergens in patients suffering from atopicdermatitis etc., in Example 13 to Example 15, dermatitis induced byrepeated inoculation of human chymase into the ears was analyzed for thepurpose of studying the effects of a repeated degranulation reaction onthe skin. Further, in Examples 16 to 17, the effects of chymase on theexpression of SCF, the major cytokine for mast cells, are shown as oneof the mechanisms of chymase-induced dermatitis. Further, in Example 18,the effects of chymase inhibitor on dermatitis was examined using NC/Ngamice as the second model induced by repeated exposure to antigen.

Preparation Example 1 Synthesis of7-chloro-3-(3-hydroxybenzenesulfonyl)-2,4(1H,3H)-quinazolinedione(Compound 1)

Following the Synthesis Method (B), 938 mg (5.42 mmol) of3-hydroxybenzenesulfonamide was dissolved in 40 ml of tetrahydrofuran,then 892 μl (5.96 mmol) of 1,8-diazabicyclo[5,4,0]-7-undecene(hereinafter referred to as DBU) was added dropwise. The reactionsolution was stirred at room temperature for 15 minutes, then 1.66 g(5.42 mmol) of methyl 4-chloro-2-N-phenoxycarbonylanthranilate was addedand the mixture was stirred at room temperature overnight. An excessamount of water was poured into the reaction solution, then the mixturewas made acidic with hydrochloric acid and extracted with ethyl acetate.The organic layer was washed with water and saturated saline, dried overanhydrous magnesium sulfate, and concentrated. The crude product thusobtained was purified by silica gel column chromatography (0% to 5%methanol/dichloromethane) to obtain 1.23 g (yield 59%) of methyl4-chloro-2-{[(3-hydroxybenzenesulfonylamino)carbonyl]amino} benzoate.Properties: colorless amorphous, PMR (δ ppm, DMSO-d₆): 3.91 (3H, s),7.02 (1H, m), 7.09 (1H, m), 7.34 (1H, t), 7.57 (2H, m), 7.89 (1H, d),8.38 (1H, d), 10.94 (1H, s). Next, the 1.23 g (3.2 mmol) of the compoundthus obtained was dissolved in 20 ml of methanol, then 10 ml of 2Nsodium hydroxide aqueous solution was added dropwise. The reactionsolution was stirred at room temperature for 15 minutes, then an excessamount of water was added and the mixture was made acidic withhydrochloric acid. This was then stirred to cause crystals toprecipitate which were then obtained by filtration and dried to obtaincarboxylic acid. The product thus obtained was dissolved in 50 ml oftetrahydrofuran (hereinafter referred to as THF), then 434 mg (2.68mmol) of CDI was added under ice cooling and the mixture was stirred for30 minutes. The reaction solution was diluted with ethyl acetate, washedwith water and saturated saline, and dried over anhydrous magnesiumsulfate, then concentrated to obtain a crude product. The crude productwas purified by silica gel column chromatography (ethylacetate:n-hexane=1:2) to obtain 230 mg (yield 20%: 2 steps) of theabove-identified compound. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 7.12 (2H, s),7.24 (1H, d), 7.48 (1H, t), 7.58 (2H, s), 7.85 (1H, d), 10.28 (1H, s),11.63 (1H, s).

Preparation Example 2 Synthesis of3-(2-aminobenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 2)

2.7 g (15.7 mmol) of 2-aminobenzenesulfonamide and 4.8 g (15.7 mmol) ofmethyl 4-chloro-2-N-phenoxycarbonylanthranilate were treated in the sameway as Preparation Example 1 to obtain 3.2 g (yield 58%: 3 steps) of theabove-identified compound. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 6.46 (2H, s),6.65 (1H, t), 6.81 (1H, d), 7.12 (1H, s), 7.23 (1H, d), 7.34 (1H, t),7.76 (1H, d), 7.86 (1H, d).

Preparation Example 3 Synthesis of7-chloro-3-(2-methylsulfonylaminobenzenesulfonyl)-2,4(1H,3H)-quinazolinedione(Compound 3)

22 mg (0.06 mmol) of Compound 2 was dissolved in 200 μl of pyridine,11.6 μl (0.15 mmol) of methanesulfonyl chloride was added dropwise, thenthe resultant mixture was stirred at room temperature overnight. Anexcess amount of water was added to the reaction solution and themixture was extracted with ethyl acetate. The organic layer was washedwith 1N aqueous hydrochloric acid solution and saturated saline, thendried over anhydrous magnesium sulfate and concentrated to obtain acrude product. The crude product was crystallized from diethyl ether toobtain 16 mg (0.04 mmol) of the above-identified compound. Properties:colorless crystal, Melting point: >200° C. (decomposition), PMR (δ ppm,DMSO-d₆): 3.61 (3H, s), 7.10 (1H, d), 7.20 (1H, d), 7.74 (1H, d),7.82-7.90 (4H, m), 8.34 (1H, d), 11.70 (1H, s).

Preparation Example 4 Synthesis of3-(4-aminobenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 4)

2.7 g (15.7 mmol) of 4-aminobenzenesulfonamide and 4.8 g (15.7 mmol) ofmethyl 4-chloro-2-N-phenoxycarbonylanthranilate were treated in the sameway as Preparation Example 1 to obtain 7.9 g (yield 94%) of methyl2-{[(4-aminobenzenesulfonylamino)carbonyl]amino}-4-chlorobenzoate.Properties: colorless amorphous, PMR (δ ppm, DMSO-d₆): 3.59 (3H, s),5.37 (2H, s), 6.45 (2H, d), 6.83 (1H, dd), 7.41 (2H, d), 7.81 (1H, d),8.66 (1H, d), 9.64 (1H, s).

Then, from the resultant 7.9 g (14.8 mmol) of sulfonylurea product, inthe same way, 4.3 g (yield 83%: 2 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 6.39 (2H, s),6.63 (2H, d), 7.09 (1H, s), 7.22 (1H, d), 7.76 (2H, d), 7.83 (1H, d),11.51 (1H, s).

Preparation Example 5 Synthesis of3-(3-carboxymethyl-benzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 5)

Following the Synthesis Method (A), 3.27 g (11.6 mmol) of3-allyloxycarbonylmethylbenzenesulfonyl isocyanate was dissolved in 100ml of anhydrous THF, then 1.98 g (11.5 mmol) of 4-chloroanthranilic acidwas added and the mixture was stirred at room temperature for 2 hours.The reaction solution was cooled with ice water, then 1.87 g (11.5 mmol)of CDI was added and the resultant mixture was stirred under ice coolingfor 30 minutes. An excess amount of water was poured into the reactionsolution, then the mixture was extracted with ethyl acetate. The organiclayer was washed, dried, and concentrated to obtain a crude product.This was crystallized with a small amount of ethyl acetate to obtain 2.0g (yield 40%) of3-(3-allyloxy-carbonylmethylbenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione.The allyl product thus obtained was dissolved in 100 ml of a formicacid-THF (1:9) mixture and 700 mg of triphenylphosphine was added. Thereactor was shaded from light and under nitrogen atmosphere, then 700 mgof tetrakis(triphenylphosphine)palladium (0) was added and the resultantmixture was stirred while shaded at room temperature overnight. Thereaction solution was concentrated in vacuo and the solid obtained waswashed with methylene chloride to obtain 1.47 g (yield 81%) of theabove-identified compound. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 3.76 (2H, s),7.13 (1H, s), 7.24 (1H, d), 7.61-7.69 (2H, m), 7.86 (1H, d), 8.05 (2H,s), 12.50 (1H, br).

Preparation Example 6 Synthesis of3-(4-carboxymethyl-benzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 6)

1.10 g (3.95 mmol) of 4-allyloxycarbonylmethyl-benzenesulfonylisocyanate and 678 mg (3.95 mmol) of 4-chloroanthranilic acid weretreated in the same way as in Preparation Example 5 to obtain 657 mg(yield 38%) of3-(4-allyloxycarbonylbenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione.538 mg (1.24 mmol) thereof was treated in the same way to obtain 342 mgof the above-identified compound (yield 70%). Properties: colorlesscrystal, Melting point: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆):3.75 (2H, s), 7.13 (1H, s), 7.23 (1H, d), 7.61-7.69 (2H, m), 7.86 (1H,d), 8.05 (2H, s), 12.07 (2H, br).

Preparation Example 7 Synthesis of(±)-2-{4-[(7-chloro-2,4(1H,3H)-quinazolin-3-yl)sulfonyl]phenyl}butyricAcid (Compound 7)

1.02 g (3.41 mmol) of t-butyl (±)-2-(4-amino-sulfonylphenyl)butyrateacid and 1.04 g (3.41 mmol) of methyl4-chloro-2-N-phenoxycarbonylanthranilate were treated in the same way asPreparation Example 1 to obtain 1.46 g (yield 84%) of methyl2-[({4-[1-(t-butoxycarbonyl)propyl]benzenesulfonylamino}carbonyl)amino]-4-chlorobenzoate.Properties: colorless amorphous, PMR (δ ppm, CDCl₃): 0.89 (3H, t), 1.38(9H, s), 1.69-1.76 (1H, m), 2.03-2.10 (1H, m), 3.42 (1H, t), 3.94 (3H,s), 7.04 (1H, d), 7.47 (2H, d), 7.93 (1H, d), 8.01 (2H, d), 8.45 (1H,br), 11.04 (1H, br).

Next, 4.3 ml (8.6 mmol) of 2N sodium hydroxide aqueous solution was usedto similarly form carboxylic acid in an amount of 1.43 g and 463 mg(2.86 mmol) of CDI was used to obtain 970 mg (yield 71%: 2 steps) oft-butyl(±)-2-{4-[(7-chloro-2,4(1H,3H)-quinazolin-3-yl)sulfonyl]phenyl}butyrate.

Further, the t-butylester thus obtained was dissolved in 5 ml ofdichloromethane, then 5 ml of trifluoroacetic acid was added and theresultant mixture was stirred at room temperature for 40 minutes. Thereaction solution was concentrated in vacuo and the resultant crudeproduct was washed with a small amount of diethyl ether to obtain 820 mgof the above-identified compound (yield 96%). Properties: colorlesscrystal, Melting point: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆):0.84 (3H, t), 1.67-1.75 (1H, m), 1.98-2.05 (1H, m), 3.62 (1H, t), 7.11(1H, s), 7.24 (1H, d), 7.61 (2H, d), 7.86 (1H, d), 8.13 (2H, d), 11.62(1H, s).

Preparation Example 8 Synthesis of3-(3-amino-4-chlorobenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 8)

1.0 g (2.93 mmol) of 3-benzyloxycarbonylamino-4-chlorobenzenesulfonamideand 1.18 g (2.93 mmol) of benzyl4-chloro-2-N-phenoxycarbonylanthranilate were treated in the same way asPreparation Example 1 to obtain 1.43 g (yield 78%) of benzyl2-{[(3-benzyloxycarbonylamino-4-chlorobenzenesulfonylamino)carbonyl]amino}-4-chlorobenzoate. Properties: colorlessamorphous, PMR (δ ppm, DMSO-d₆): 5.19 (2H, s), 5.36 (2H, s), 7.21 (1H,dd), 7.34-7.48 (10H, m), 7.72-7.76 (2H, m), 7.97 (1H, d), 8.25 (1H, d),8.30 (1H, d), 9.53 (1H, s), 10.30 (1H, s). 1.38 g (2.20 mmol) thereofwas dissolved in 50 ml of THF, then 200 mg of palladium-carbon (10%) wasadded and the mixture was stirred under a hydrogen flow for 2 hours. Thereaction mixture was filtered with Celite to remove thepalladium-carbon, then the filtrate was concentrated in vacuo to obtaina carboxylic acid. The product obtained was suspended in 50 ml of THF,then 356 mg (2.20 mmol) of CDI was added under ice cooling and theresultant mixture was treated in the same way as Preparation Example 1to obtain 560 mg (yield 66%: 2 steps) of the above-identified compound.Properties: colorless crystal, Melting point: >200° C. (decomposition),PMR (δ ppm, DMSO-d₆): 6.00 (2H, s), 7.12 (1H, s), 7.26 (2H, t), 7.48(1H, d), 7.66 (1H, s), 7.86 (1H, d), 11.76 (1H, br).

Preparation Example 9 Synthesis of3-(4-amino-3,5-dichlorobenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 9)

1.06 g (4.40 mmol) of 4-amino-3,5-dichloro-benzenesulfonamide and 1.34 g(4.40 mmol) of methyl 4-chloro-2-N-phenoxycarbonylanthranilate weretreated in the same way as Preparation Example 1 to obtain 905 mg (yield44%) of methyl2-{[(4-amino-3,5-dichlorobenzenesulfonylamino)carbonyl]amino}-4-chlorobenzoate.Properties: colorless amorphous, PMR (δ ppm, DMSO-d₆): 3.87 (3H, s),6.59 (2H, br), 7.22 (1H, dd), 7.72 (2H, s), 7.93 (1H, d), 8.24 (1H, d),10.17 (1H, s).

Then, from 905 mg (2.0 mmol) of the resultant sulfonylurea product, inthe same way, 660 mg (yield 82%: 2 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (6 ppm, DMSO-d₆): 6.80 (2H, s),7.12 (1H, s), 7.24 (1H, d), 7.86 (1H, d), 7.92 (2H, s), 11.63 (1H, br).

Preparation Example 10 Synthesis of3-(3-amino-4-methylbenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 10)

960 mg (3.00 mmol) of3-benzyloxycarbonylamino-4-methylbenzenesulfonamide and 1.14 g (3.00mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate were treated inthe same way as in Preparation Example 8 to obtain 1.14 g (yield 62% ofbenzyl2-{[(3-benzyloxycarbonylamino-4-methylbenzenesulfonylamino)carbonyl]amino}-4-chlorobenzoate.Properties: colorless amorphous, PMR (δ ppm, DMSO-d₆): 2.30 (3H, s),5.17 (2H, s), 5.36 (2H, s), 7.20 (1H, dd), 7.33-7.48 (11H, m), 7.63 (1H,d), 7.97 (1H, d), 8.11 (1H, s), 8.25 (1H, s), 9.27 (1H, s), 10.30 (1H,s), 12.20 (1H, br).

Then, from 1.14 g (1.87 mmol) of the resultant sulfonylurea product, inthe same way, 190 mg (yield 27%: 2 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 2.12 (3H, s),5.47 (2H, s), 7.12 (1H, s), 7.16-7.25 (3H, m), 7.38 (1H, s), 7.85 (1H,d), 11.58 (1H, s).

Preparation Example 11 Synthesis of3-[(3-carboxymethylaminophenyl)sulfonyl]-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 11)

1.62 g (5.65 mmol) of 3-t-butoxycarbonyl-methylaminobenzenesulfonamideand 1.73 g (5.65 mmol) of methyl4-chloro-2-N-phenoxycarbonylanthranilate were treated in the same way asin Preparation Example 7 to obtain 209 mg (yield 9%: 4 steps) of theabove-identified compound. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 3.86 (2H, s),6.88 (1H, s), 7.12 (1H, s), 7.24 (1H, d), 7.30-7.38 (3H, m), 7.86 (1H,d), 11.61 (1H, br).

Preparation Example 12 Synthesis of3-(3-aminobenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 12)

3.5 g (12.9 mmol) of 3-t-butoxycarbonylamino-benzenesulfonamide and 3.9g (12.8 mmol) of methyl 4-chloro-2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 7 to obtain 2.2 g(yield 49%: 4 steps) of the above-identified compound. Properties:colorless crystal, Melting point: >200° C. (decomposition), PMR (δ ppm,DMSO-d₆): 5.72 (2H, s), 6.87 (1H, d), 7.12 (1H, s), 7.23-7.27 (2H, m),7.33 (1H, s), 7.86 (1H, d), 11.61 (1H, s).

Preparation Example 13 Synthesis of2-{3-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]phenylaminocarbonyl}propionicAcid (Compound 13)

100 mg (0.28 mmol) of Compound 12 was dissolved in 5 ml of THF, 100 mg(1.0 mmol) of succinic anhydride was added, and the resultant mixturewas heated and refluxed for 3 hours. The reaction solution wasconcentrated in vacuo and the crude product thus obtained wascrystallized with ethyl acetate-diethyl ether to obtain 120 mg (yield96%) of the above-identified compound. Properties: colorless crystal,Melting point: 187-188° C., PMR (δ ppm, DMSO-d₆): 2.54 (2H, d), 2.59(2H, d), 7.12 (1H, s), 7.24 (1H, d), 7.59 (1H, t), 7.80 (1H, d), 7.86(1H, d), 7.96 (1H, d), 8.41 (1H, s), 10.40 (1H, s), 11.63 (1H, br),12.10 (1H, br).

Preparation Example 14 Synthesis of3-{3-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]phenyl}acrylicacid (Compound 14)

1.54 g (5.44 mmol) of t-butyl 3-(3-aminosulfonyl)phenylacrylate and 1.66g (5.44 mmol) of methyl 4-chloro-2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 7 to obtain 2.18 g(yield 81%) of methyl2-({[3-(3-t-butoxy-3-oxo-1-propenyl)benzenesulfonylamino]carbonyl}amino)-4-chlorobenzoate.Properties: colorless amorphous, PMR (δ ppm, CDCl₃): 1.53 (9H, s), 3.95(3H, s), 6.46 (1H, d), 7.05 (1H, d), 7.55 (1H, m), 7.57 (1H, d), 7.72(1H, m), 7.93 (1H, m), 8.04 (1H, m), 8.27 (1H, s), 8.46 (1H, d), 11.05(1H, br).

Then, from 2.18 g (4.4 mmol) of the resultant sulfonylurea product, inthe same way, 698 mg (yield 37%: 3 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 6.65 (1H, d),7.12 (1H, s), 7.25 (1H, d), 7.69 (1H, d), 7.72 (1H, t), 7.87 (1H, d),8.12 (2H, q), 8.37 (1H, s), 11.64 (1H, s).

Preparation Example 15 Synthesis of4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]salicylic acid(Compound 15)

1.0 g (3.66 mmol) of 4-t-butoxycarbonyl-3-hydroxybenzenesulfonamide and1.12 g (3.66 mmol) of methyl 4-chloro-2-N-phenoxycarbonylanthranilatewere treated in the same way as in Preparation Example 7 to obtain 1.79g (yield 100%) of methyl2-{[(4-t-butoxycarbonyl-3-hydroxybenzenesulfonylamino)carbonyl]amino}-4-chlorobenzoate.Properties: colorless amorphous, PMR (δ ppm, DMSO-d₆): 1.57 (9H, s),3.87 (3H, s), 7.14 (1H, d), 7.40-7.45 (2H, m), 7.85 (1H, d), 7.92 (1H,d), 8.32 (1H, d), 10.13 (1H, s), 10.82 (1H, s).

Then, from 1.78 g (3.66 mmol) of the resultant sulfonylurea product, inthe same way, 370 mg (yield 25%: 3 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 7.13 (1H, s),7.26 (1H, d), 7.69 (1H, d), 7.87 (1H, d), 8.01 (1H, d), 11.67 (1H, s).

Preparation Example 16 Synthesis of4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]salicylic AcidMonosodium Salt (Compound 16)

50 mg (0.13 mmol) of Compound 15 was suspended in approximately 1 ml ofTHF, then 126 μl of 1N sodium hydroxide aqueous solution was addeddropwise. The solution was confirmed to have become uniform, then 30 mlof water was added and the mixture freeze-dried to quantitatively obtainthe above-identified compound in an amorphous state in an amount of 52mg. Properties: colorless amorphous, PMR (δ ppm, CD₃OD): 7.11 (1H, s),7.19 (1H, d), 7.58 (1H, d), 7.63 (1H, s), 7.92 (1H, d), 8.03 (1H, d).

Preparation Example 17 Synthesis of4-[(7-chloro-1-2,4(1H,3H-quinazolinedion-3-yl)sulfonyl]anthranilic Acid(Compound 17)

2.84 g (6.99 mmol) of3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonamide and 2.67 g(6.99 mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 8 to obtain 3.74 g(yield 77%) of benzyl2-{[(3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonylamino)carbonyl]amino}-4-chlorobenzoate.Properties: colorless amorphous, PMR (δ ppm, DMSO-d₆): 1.54 (9H, s),5.19 (2H, s), 5.34 (2H, s), 7.05 (1H, m), 7.34-7.58 (10H, m), 7.60 (1H,d), 7.90 (1H, d), 7.98 (1H, d), 8.50 (1H, br), 8.62 (1H, s), 10.00 (1H,br), 10.41 (1H, s).

Then, from 3.74 g (5.39 mmol) of the resultant sulfonylurea, in the sameway, 690 mg (yield 30%: 2 steps) of t-butyl4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilate wasobtained, then this was subjected to a similar debutylation reaction toobtain 503 mg (yield 84%) of the above-identified compound. Properties:colorless crystal, Melting point: >200° C. (decomposition), PMR (δ ppm,DMSO-d₆): 7.14 (1H, s), 7.18 (1H, d), 7.25 (1H, d), 7.59 (1H, s), 7.87(1H, d), 7.89 (1H, d), 11.62 (1H, s).

Preparation Example 18 Synthesis of4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilic AcidMonosodium Salt (Compound 18)

50 mg (0.13 mmol) of Compound 17 was suspended in approximately 1 ml ofTHF, then 126 μl of 1N sodium hydroxide aqueous solution was addeddropwise. The solution was confirmed to have become uniform, then 30 mlof water was added and the mixture was freeze-dried to quantitativelyobtain the above-identified compound in an amorphous state in an amountof 52 mg. Properties: colorless amorphous, PMR (δ ppm, DMSO-d₆):7.11-7.22 (3H, m), 7.37 (1H, s), 7.83 (1H, d), 7.91 (1H, d).

Preparation Example 19 Synthesis of3-(4-hydroxybenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 19)

1.50 g (7.03 mmol) of 4-allyloxybenzenesulfonyl isocyanate and 1.2 g(7.03 mmol) of 4-chloroanthranilic acid were treated in the same way asin Preparation Example 5 to obtain 1.5 g (yield 53%) of3-(4-allyloxybenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione. 500mg (1.27 mmol) thereof was similarly treated to obtain 405 mg of theabove-identified compound (yield 90%). Properties: colorless crystal,Melting point: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 6.98 (2H,d), 7.11 (1H, s), 7.23 (1H, d), 7.85 (1H, d), 8.00 (2H, d), 11.25 (1H,br).

Preparation Example 20 Synthesis of4-[(2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]salicylic Acid (Compound20)

618 mg (2.26 mmol) of 4-t-butoxycarbonyl-3-hydroxybenzenesulfonamide and613 mg (2.26 mmol) of methyl 2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 17 to obtain 792 mg(yield 78%) of methyl2-{[(4-t-butoxycarbonyl-3-hydroxybenzene-sulfonylamino)carbonyl]amino}benzoate.Properties: colorless amorphous, PMR (δ ppm, CDCl₃): 1.60 (9H, s), 3.97(3H, s), 7.09 (1H, t), 7.49-7.52 (2H, m), 7.65 (1H, d), 7.90 (1H, d),8.01 (1H, dd), 8.33 (1H, d), 10.98 (1H, s), 11.18 (1H, s).

Then, from 790 mg (1.75 mmol) of the resultant sulfonylurea product, inthe same way, 100 mg (yield 8%: 3 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 7.13 (1H, d),7.22 (1H, t), 7.63-7.69 (3H, m), 7.87 (1H, d), 8.01 (1H, d), 11.57 (1H,s).

Preparation Example 21 Synthesis of5-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]salicylic Acid(Compound 21)

320 mg (1.17 mmol) of 3-t-butoxycarbonyl-4-hydroxybenzenesulfonamide and447 mg (1.17 mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilatewere treated in the same way as in Preparation Example 17 to obtain 611mg (yield 93%) of benzyl2-{[(3-t-butoxycarbonyl-4-hydroxybenzenesulfonylamino)carbonyl]amino}-4-chlorobenzoate.Properties: colorless amorphous, PMR (δ ppm, CDCl₃): 1.62 (9H, s), 5.35(2H, s), 7.01-7.05 (2H, m), 7.37-7.41 (5H, m), 7.96 (1H, d), 8.10 (1H,dd), 8.46-8.48 (2H, m), 10.99 (1H, s), 11.66 (1H, s).

Then, from 611 mg (1.09 mmol) of the resultant sulfonylurea product, inthe same way, 114 mg (yield 33%: 3 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 7.11 (1H, s),7.19 (1H, d), 7.24 (1H, d), 7.86 (1H, d), 8.20 (1H, d), 8.56 (1H, s),11.57 (1H, s).

Preparation Example 22 Synthesis of3-(3-acetamide-4-methoxybenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione (Compound 22)

500 mg (2.19 mmol) of 3-acetamide-4-methoxybenzenesulfonamide and 836 mg(2.19 mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 8 to obtain 812 mg(yield 70%) of benzyl2-{[(3-acetylamino-4-methoxybenzenesulfonylamino)carbonyl]amino}-4-chlorobenzoate.Properties: colorless amorphous, PMR (δ ppm, DMSO-d₆): 2.12 (3H, s),3.93 (3H, s), 5.36 (2H, s), 7.20 (1H, d), 7.24 (1H, d), 7.36-7.48 (5H,m), 7.69 (1H, d), 7.96 (1H, d), 8.24 (1H, s), 8.67 (1H, s), 9.39 (1H,s), 10.25 (1H, s), 12.11 (1H, br).

Then, from 611 mg (1.09 mmol) of the resultant sulfonylurea product, inthe same way, 250 mg (yield 39%: 2 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 2.12 (3H, s),3.95 (3H, s), 7.12 (1H, s), 7.23 (1H, d), 7.30 (1H, d), 7.85 (1H, d),7.89 (1H, d), 8.80 (1H, s), 9.42 (1H, s), 11.59 (1H, br).

Preparation Example 23 Synthesis of3-(3-amino-4-methoxybenzenesulfonyl)-7-chloro-2,4(1H,3H)-quinazolinedione(Compound 23)

400 mg (1.40 mmol) of3-t-butoxycarbonylamino-4-methoxybenzenesulfonamide and 533 mg (1.40mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate were treated inthe same way as in Preparation Example 17 to obtain 86 mg (yield 16%: 4steps) of the above-identified compound. Properties: colorless crystal,Melting point: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 3.81 (3H,s), 7.26-7.37 (5H, m), 7.77 (1H, s), 7.90 (1H, d), 7.94 (1H, d), 11.73(1H, s).

Preparation Example 24 Synthesis of7-chloro-3-(4-methoxy-3-methylsulfonylaminobenzenesulfonyl)-2,4(1H,3H)-quinazolinedione(Compound 24)

500 mg (1.89 mmol) of 4-methoxy-3-methylsulfonylaminobenzenesulfonamideand 722 mg (1.89 mmol) of benzyl4-chloro-2-N-phenoxycarbonylanthranilate were treated in the same way asin Preparation Example 8 to obtain 888 mg (yield 83%) of benzyl2-({[(4-methoxy-3-methylsulfonylamino)benzenesulfonylamino]carbonyl}amino)-4-chlorobenzoate. Properties: colorlessamorphous, PMR (δ ppm, DMSO-d₆): 2.12 (3H, s), 3.93 (3H, s), 5.36 (2H,s), 7.20 (1H, d), 7.24 (1H, d), 7.36-7.48 (5H, m), 7.69 (1H, d), 7.96(1H, d), 8.24 (1H, s), 8.67 (1H, s), 9.39 (1H,'s), 10.25 (1H, s), 12.11(1H, br).

Then, from 880 mg (1.55 mmol) of the resultant sulfonylurea product, inthe same way, 620 mg (yield 85%: 2 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 3.04 (3H, s),3.94 (3H, s), 7.11 (1H, s), 7.23 (1H, d), 7.34 (1H, d), 7.86 (1H, d),7.99 (1H, d), 8.10 (1H, s).

Preparation Example 25 Synthesis of4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-ylsulfonyl]-1-hydroxy-naphthalene-2-carboxylic Acid (Compound 25)

323 mg (1.00 mmol) of3-t-butoxycarbonyl-4-hydroxy-1-naphthalenesulfonamide and 381 mg (1.00mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate were treated inthe same way as in Preparation Example 17 to obtain 447 mg (yield 73%)of4-({[(2-benzyloxycarbonyl-5-chloroanilino)carbonyl]amino}sulfonyl)-1-hydroxy-2-naphthalenecarboxylicacid t-butyl ester. Properties: colorless amorphous, PMR (δ ppm,DMSO-d₆): 1.66 (9H, s), 5.34 (3H, s), 6.98 (1H, d), 7.35-7.48 (5H, m),7.66 (1H, m), 7.81 (1H, m), 7.89 (1H, d), 8.37 (2H, m), 8.44 (1H, s),8.71 (1H, d), 10.02 (1H, br), 12.52 (1H, br).

Then, from 445 mg (0.72 mmol) of the resultant sulfonylurea product, inthe same way, 56 mg (yield 18%: 3 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 7.08 (1H, s),7.20 (1H, d), 7.63 (1H, t), 7.77 (1H, t), 7.84 (1H, d), 8.42 (1H, d),8.51 (1H, d), 8.75 (1H, s), 11.57 (1H, s).

Preparation Example 26 Synthesis of5-[(7-chloro-2.4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilic Acid(Compound 26)

834 mg (2.05 mmol) of4-benzyloxycarbonylamino-3-t-butoxycarbonylbenzenesulfonamide and 783 mg(2.05 mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 17 to obtain 1.18 g(yield 83%) of benzyl2-{[(4-benzyloxycarbonylamino-3-t-butoxycarbonylbenzenesulfonylamino)carbonyl]amino}-4-chlorobenzoate.Properties: colorless amorphous, PMR (δ ppm, CDCl₃): 1.56 (9H, s), 5.22(2H, s), 5.37 (2H, s), 7.04 (1H, dd), 7.33-7.42 (10H, m), 7.97 (1H, d),8.14 (1H, d), 8.45 (1H, d), 8.60 (1H, d), 8.65 (1H, d), 11.01 (1H, s),11.11 (1H, s).

Then, from 1.17 g (1.69 mmol) of the resultant sulfonylurea product, inthe same way, 404 mg (yield 60%: 3 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 6.89 (1H, d),7.11 (1H, s), 7.23 (1H, d), 7.85 (1H, d), 7.98 (1H, d), 8.51 (1H, s),11.51 (1H, s).

Preparation Example 27 Synthesis of4-[(7-methoxy-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilic Acid(Compound 27)

500 mg (1.23 mmol) of3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonamide and 460 mg(1.22 mmol) of benzyl 4-methoxy-2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 17 to obtain 15 mg(yield 3.1%: 4 steps) of the above-identified compound. Properties:colorless crystal, Melting point: >200° C. (decomposition), PMR (δ ppm,DMSO-d₆): 3.82 (3H, s), 6.58 (1H, s), 6.80 (1H, d), 7.16 (1H, d), 7.56(1H, s), 7.80 (1H, d), 7.90 (1H, d), 11.49 (1H, s).

Preparation Example 28 Synthesis of(±)-7-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-2-oxo-1H,3H-quinoline-3-carboxylicacid (Compound 28)

400 mg (1.23 mmol) of(±)-3-t-butoxycarbonyl-2-oxo-1H,3H-quinoline-7-sulfonamide and 468 mg(1.23 mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 17 to obtain 649 mg(yield 86%) of8-({[(2-benzyloxycarbonyl-5-chloroanilino)carbonyl]amino}sulfonyl)-2-oxo-1,2,3,4-tetrahydro-3-quinolinecarboxylic acid t-butyl ester. Properties: colorless amorphous, PMR (6ppm, CDCl₃): 1.32 (9H, s), 3.18-3.30 (2H, m), 3.54 (1H, m), 5.35 (2H,s), 6.85 (1H, m), 7.00 (1H, m), 7.35-7.39 (5H, m), 7.87-7.96 (3H, m),8.47 (1H, m), 8.78 (1H, br), 10.92 (1H, br).

Then, from 640 mg (1.04 mmol) of the resultant sulfonylurea product, inthe same way, 258 mg (yield 55%: 3 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (6 ppm, DMSO-d₆): 3.23-3.31 (2H,m), 3.59 (1H, t), 7.07 (1H, d), 7.12 (1H, s), 7.25 (1H, d), 7.86 (1H,d), 7.96 (1H, d), 7.98 (1H, d), 10.84 (1H, s), 11.60 (1H, s).

Preparation Example 29 Synthesis of(±)-6-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-3-oxo-1,4-benzoxazine-2-carboxylicAcid (Compound 29)

300 mg (0.91 mmol) of(±)-2-t-butoxycarbonyl-3-oxo-1,4-benzoxazin-6-sulfonamide and 349 mg(0.91 mmol) of benzyl 4-chloro-2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 17 to obtain 417 mg(yield 74%) of5-({[(2-benzyloxycarbonyl-5-chloroanilino)carbonyl]amino}sulfonyl)-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylicacid t-butyl ester. Properties: colorless amorphous, PMR (6 ppm,DMSO-d₆): 1.29 (9H, s), 5.37 (2H, s), 5.42 (2H, s), 7.19-7.26 (2H, m),7.37-7.57 (7H, m), 7.97 (1H, d), 8.25 (1H, d), 10.27 (1H, s), 11.25 (1H,s), 12.22 (1H, br).

Then, from 417 mg (0.68 mmol) of the resultant sulfonylurea product, inthe same way, 100 mg (yield 32%: 3 steps) of the above-identifiedcompound was obtained. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (6 ppm, DMSO-d₆): 5.47 (1H, s),7.11 (1H, s), 7.24 (1H, d), 7.29 (1H, d), 7.76 (1H, s), 7.78 (1H, d),7.86 (1H, d), 11.25 (1H, s), 11.62 (1H, s).

Preparation Example 30 Synthesis of4-[(7-hydroxy-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilic Acid(Compound 30)

620 mg (1.53 mmol) of3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonamide and 550 mg(1.51 mmol) of benzyl 4-hydroxy-2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 17 to obtain 25 mg(yield 4%: 4 steps) of the above-identified compound. Properties:colorless crystal, Melting point: >200° C. (decomposition), PMR (δ ppm,DMSO-d₆): 6.48 (1H, s), 6.61 (1H, d), 7.14 (1H, d), 7.51 (1H, s), 7.70(1H, d), 7.90 (1H, d), 10.80 (1H, s), 11.39 (1H, s).

Preparation Example 31 Synthesis of4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-2-N-propionylanthranilicAcid (Compound 31)

840 mg (1.86 mmol) of Compound 17 was dissolved in 8 ml of 1,4-dioxane,240 μl (2.79 mmol) of propionyl chloride was added dropwise, then theresultant mixture was stirred overnight at 60° C. An excess of water wasadded to the reaction solution and the mixture was extracted with ethylacetate. The organic layer thus obtained was washed, dried, andconcentrated to obtain a crude product of t-butyl4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-2-N-propionylanthranilate.The obtained crude product was stirred at room temperature in 3 ml oftrifluoroacetic acid for 1 hour, then the reaction solution wasconcentrated in vacuo to obtain a crude product. This was washed bydiethyl ether to obtain 400 mg (yield 48%: 2 steps) of theabove-identified compound. Properties: colorless crystal, Meltingpoint: >200° C. (decomposition), PMR (δ ppm, DMSO-d₆): 1.10 (3H, t),2.45 (2H, dd), 7.11 (1H, s), 7.24 (1H, d), 7.85 (1H, d), 7.88 (1H, d),8.17 (1H, d), 9.18 (1H, s), 11.07 (1H, s), 11.63 (1H, s).

Preparation Example 32 Synthesis of4-[(6-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]anthranilic Acid(Compound 32)

300 mg (0.74 mmol) of3-benzyloxycarbonylamino-4-t-butoxycarbonylbenzenesulfonamide and 310 mg(0.81 mmol) of benzyl 5-chloro-2-N-phenoxycarbonylanthranilate weretreated in the same way as in Preparation Example 17 to obtain 75 mg(yield 26%: 4 steps) of the above-identified compound. Properties:colorless crystal, Melting point: >200° C. (decomposition), PMR (δ ppm,DMSO-d₆): 7.13-7.20 (2H, m), 7.56 (1H, s), 7.72 (1H, d), 7.82 (1H, s),7.90 (1H, d), 11.68 (1H, s).

Preparation Example 33 Synthesis of4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-2-N-methanesulfonylanthranilicAcid (Compound 33)

200 mg (0.44 mmol) of Compound 17 was treated in the same way as inPreparation Example 3 to obtain 81 mg of t-butyl4-[(7-chloro-2,4(1H,3H)-quinazolinedion-3-yl)sulfonyl]-2-N-methanesulfonylanthranilate.This was used to perform the same debutylation reaction to obtain 53 mg(yield 25%: 2 steps) of the above-identified compound. Properties:colorless crystal, Melting point: >200° C. (decomposition), PMR (δ ppm,DMSO-d₆): 3.24 (3H, s), 7.11 (1H, s), 7.25 (1H, d), 7.85-7.91 (2H, m),8.23 (1H, d), 8.39 (1H, s), 11.05 (1H, br), 11.70 (1H, s).

Preparation Example 34 Synthesis of3-(3-aminobenzenesulfonyl)-7-chloro-2,4-(1H,3H)quinazolinedionMethanesulfonic Acid Salt (Compound 34) 2.15 g (6.10 mmol) of compound12 was dissolved in

65 ml of THF and 0.4 ml of methanesulfonic acid was added dropwise. Tothis solution, 200 ml of ether was added and the resultant precipate wasfiltered to obtain 2.59 g (yield 95%) of the above-identified compound.Properties: colorless amorphous, PMR (δ ppm, DMSO-d₆): 2.35 (3H, s),6.98 (1H, d), 7.12 (1H, m), 7.25 (1H, m), 7.34 (2H, s), 7.43 (1H, m),7.86 (1H, s), 11.64 (1H, s).

Preparation Example 35 Synthesis of7-chloro-3-[4-(pyrazol-3-yl)benzenesulfonyl]-2,4(1H,3H)-quinazolinedionehydrochloride (Compound 35)

Following the Synthesis Method (B), 5.65 g (25.34 mmol) of4-(pyrazol-3-yl)beneznesulfonamide was dissolved in 60 ml of THF, then7.8 ml (52.16 mmol) of DBU was added dropwise. The reaction solution wasstirred at room temperature for 10 minutes, then added with 8.5 g (27.86mmol) of methyl 4-chloro-2-phenoxycarbonyl-aminobenzoate and stirred atroom temperature for 3 hours. The reaction solution was further addedwith 400 mg (0.131 mmol) of methyl4-chloro-2-phenoxycarbonylaminobenzoate and then stirred at roomtemperature for 2 hours. An excess of an aqueous solution of citric acidwas added to the reaction solution, then extraction was performed usingethyl acetate. The organic layer was washed by water and saturatedsaline, then dried over anhydrous sodium sulfate and condensed. Methanolwas added to the condensed residue, then the mixture was stirred and theresultant crystals were obtained by filtration to obtain 10.49 g of acrude product.

10.49 g of the crude product obtained was suspended in 45 ml ofmethanol, then 90 ml of a 1N sodium hydroxide aqueous solution wasadded. The reaction solution was stirred at 60° C. for 40 minutes, thenthe precipitate was removed by filtration. The filtrate was concentratedin vacuo and the methanol distilled off, then the obtained aqueousmixture was washed by ethyl acetate. The aqueous layer was made acidicby hydrochloric acid to cause the precipitation of crystals. These werethen obtained by filtration. The filtrate was extracted by ethylacetate, the organic layer was washed by saturated saline, and theresult was dried and condensed over anhydrous sodium sulfate. Thecondensed residue and the crystals obtained by filtration above werecombined and recrystallized from THF-ethyl acetate-hexane to obtain 7.70g (yield of 72% in two steps) ofN-[4-(pyrazol-3-yl)benzenesulfonyl]-N′-(2-carboxyl-5-chlorophenyl)urea(properties: colorless crystal, melting point: 129 to 132° C., PMR (δppm, DMSO-d₆): 6.81 (1H, d), 7.02 (1H, dd), 7.78 (1H, s), 7.89-7.92 (3H,m), 7.96 (2H, d), 8.24 (1H, s), 10.57 (1H, br).

3.0 g (7.14 mmol) of the urea derivative obtained above was dissolved in60 ml of THF. 1.2 g (7.40 mmol) of CDI was then added under ice coolingand the result stirred for 2 hours. The reaction solution was diluted byethyl acetate, then successively washed by a citric acid aqueoussolution, saturated saline, a 0.5M sodium hydrogencarbonate aqueoussolution, and saturated saline. The organic layer was dried overanhydrous sodium sulfate, then condensed to obtain a crude product. Thecrude product was recrystallized from ethyl acetate to obtain 1.93 g(yield: 67%) of 7-chloro-3-[4-(pyrazol-3-yl)benzenesulfonyl]-2,4(1H,3H)-quinazolinedione (properties: colorlesscrystal, melting point: 124 to 126° C. (decomposition), PMR (δ ppm,CDCl₃-CD₃OD): 6.73 (1H, s), 7.09 (1H, s), 7.16 (2H, d), 7.48 (1H, s),7.66 (1H, s), 7.9-8.1 (3H, m), 8.32 (2H, d).

545 mg (1.35 mmol) of the quinazoline derivative obtained above wasdissolved in 35 ml of THF, then 0.4 ml of a 1,4-dioxane solution of 4Mhydrochloric acid was added dropwise. 20 ml of ether was added to thissolution, then the precipitated crystal was obtained by filtration toobtain 572 mg (yield: 96%) of the above-referenced compound. Properties:colorless crystal, melting point: >200° C. (decomposition), PMR (δ ppm,DMSO-d₆): 6.91 (1H, d), 7.15 (1H, d), 7.24 (1H, dd), 7.84 (1H, d), 7.86(1H, d), 8.01 (2H, d), 8.17 (2H, d), 11.7 (1H, s).

Example 1 Measurement of Chymase Inhibitory Activity

Human heart chymase was purified according to the method of Urata et al.(J. Biol. Chem., 1990, 265, 22348). The inhibitory activity of thequinazoline derivatives of the present invention with respect to chymasewas measured in the following manner. That is, the purified enzymesolution was diluted to a suitable concentration with 0.1Mtris-hydrochloride buffer (pH=7.5), 1M sodium chloride, and 0.01%TritonX-100 to obtain an enzyme solution. A 10 mM dimethyl sulfoxide(hereinafter referred to as DMSO) solution of Suc-Ala-Ala-Pro-Phe-MCA(Peptide Institute) was diluted 20-fold at the time of use by 0.1Mtris-hydrochlorate, 1M sodium chloride, and 0.01% TritonX-100 to obtainthe substrate solution.

75 μl of the enzyme solution warmed to 30° C. was mixed with 5 μl ofDMSO solution of the test sample. The mixture was preincubated at 30° C.for 10 minutes. Next, 20 μl of a substrate solution warmed to 30° C. wasmixed with the test sample-enzyme mixture and incubated at 30° C. After10 minutes, 50 μl of 30% acetic acid was added to stop the enzymaticreaction. The amount of the AMC produced was quantified using afluorescent photometer. At the same time, a blind test was carried outby adding, instead of the test sample solution, 5 μl of DMSO andperforming the same reaction. The chymase inhibitory activity wasexpressed by a rate of inhibition, that is, the 50% inhibitionconcentration (IC₅₀), based on the blind test value.

The quinazoline derivatives of the present invention all stronglyinhibited human chymase at concentrations of 100 μM. The IC₅, values fortypical compounds are shown in Table I.

TABLE 1 Example No. IC₅₀ value (μM) 1 0.36 2 0.14 8 0.035 10 0.17 120.44 13 0.3 16 0.84 17 0.14 18 0.14 21 0.34 22 0.3 24 0.32 27 4.0 29 1.732 1.5 34 0.36

Example 2 Time-Course of Skin Reaction in Ascaris-Induced Mouse BiphasicDermatitis Model

Ascaris-induced biphasic dermatitis was induced according to the methoddescribed previously (Folia Pharmacol. Jap. 112, 221, 1998). That is,8-week old BALB/c mice (Charles River Japan) were sensitized byintraperitoneal injection of 0.5 ml of a 1:1 mixture of ascaris extract(800 μg/ml, Cosmo Bio Co., Ltd.) and an alum saline suspension (16 mg/mlin saline). Two weeks after the sensitization, 10 μl of ascaris extract(1 mg/ml) was injected intradermally to the right ear of mice. The edemainduced at the ear were evaluated immediately before intradermalinjection of the ascaris extract (n=3) and 1 hour after (n=4), 2 hoursafter (n=4), 4 hours after (n=4), 6 hours after (n=4), 16 hours after(n=4), and 24 hours after injection (n=4), by weighing ear biopsyprepared with a punch (a diameter of 6 mm, Fukui Kiko Shokai) andmeasuring their weights. The edema (mg) was expressed as the differencein the weight of the ear punch biopsy between the right and the leftears of the same mouse.

Biphasic dermatitis was induced by intradermal administration of ascarisextract to the ears of mice sensitized by the same antigen (FIG. 1). Thefirst reaction reached its peak after 1 hour, while the second reactionreached its peak after 16 hours.

Example 3 Effects of Chymase Inhibitor in Ascaris-Induced Mouse BiphasicDermatitis Model

Dermatitis was induced in accordance with the method described inExample 2 and the ear edema was measured in the same way as in Example2, 1 hour (n=6) and 16 hours (n=8) after the intradermal administrationof ascaris extract to the ears to investigate the effects of the testsubstance on dermatitis. As the chymase inhibitor, Compound 34 was used.As the control drug, diphenhydrazine (antihistamine, Sigma) andprednisolone (steroid, Nakarai Tesc Co.) were used. Each drug understudy was suspended in saline containing 0.5% hydroxypropyl celluloseand administered intraperiotoneally 60 minutes before intradermaladministration of ascaris extract. A group of mice sensitized withascaris extract and challenged by intradermal injection of saline wasused as a control (n=3).

Results

As a result of the intraperiotoneal administration of the chymaseinhibitor (Compound 34), the reaction after 1 hour (early-phasereaction) and reaction after 16 hours (late-phase reaction) of thebiphasic dermatitis induced by ascaris extract were both suppressed in adose-dependent manner. A statistically significant difference wasobserved in the dosage of 50 mg/kg (FIG. 2A). The rate of suppression at50 mg/kg was about 41% for the early-phase reaction and about 45% forthe late-phase reaction (both p<0.01, Dunnett's test). Prednisolone,which is effective against atopic dermatitis, was substantiallyineffective against the early-phase reaction, but strongly inhibited thelate-phase reaction in a dosage of 30 mg/kg (rate of suppression: 67%)(FIG. 2B). On the other hand, diphenhydrazine significantly suppressedthe early-phase reaction (rate of suppression: 79%), but exhibitedalmost no effect against the late-phase reaction (FIG. 2C).

The fact that a chymase inhibitor exhibits a suppressive action in anallergic dermatitis model exhibiting biphasic skin reaction shows theinvolvement of chymase in allergic biphasic dermatitis and usefulness ofa chymase inhibitor for such dermatitis. In particular, the finding thata chymase inhibitor, like a steroid, significantly suppresses late-phasereaction, in which antihistamines and anti-allergic agents exhibitlittle effect, shows the usefulness of a chymase inhibitor in atopicdermatitis. In the following Examples 4 to 6, the skin conditionsinduced by inoculation of human chymase into the ears of mice wereanalyzed for the purpose of further confirming the importance of chymasein a biphasic skin reaction.

Example 4 Ability of Single Administration of Human Chymase to InduceDermatitis

Recombinant human chymase was used in this Example.

Recombinant human chymase was obtained by expression and purification inaccordance with the already reported method of production of serineprotease (Biochem. Biophys. Acta 1350, 11, 1997). That is, first, cDNA(79-756) encoding mature human chymase (J. Biol. Chem. 266, 17173, 1991)was amplified by the PCR method. The PCR product was cloned to the pDEvector along with the signal sequence of human trypsin II and the regionincluding the cleaved site of enterokinase (23 amino acid). Theconstructed human chymase expression plasmid was transfected to CHOdhfr⁻cells, and the transfectants were selected by an already reported method(Arch. Biochem. Biophys. 307, 133, 1993). The fused protein of the humanchymase and trypsin secreted in the culture supernatent of the obtainedcells was concentrated using a HiTrap Heparin column (Amersham PharmaciaBiotech), then cleaved with enterokinase (Invitrogen) to produce humanmature chymase. The human mature chymase was purified using a heparin5PW column (Tosoh Corp.) In SDS-polyacrylamide gel electrophoresisanalysis, the purified chymase showed a 33-36 kDa broad band. Further,chymase activity was measured in a 0.1M Tris/HCl buffer (pH8.0) by using1 mM Suc-Ala-Ala-Pro-Phe-MCA (Peplide Institute) as a substrate andmeasuring the intensity of fluorescense of the free MCA. As a result, itwas confirmed that the purified chymase certainly has the enzymaticactivity.

Next, 20 μl of the above recombinant human chymase (hereinafter calledhuman chymase) (0.1 mg/ml) was administered intradermally to an ear ofBALB/c mice (Japan Charles River) and the time-course of the edematousreaction of the ears measured by the method described in Example 2 forthe purpose of investigating the role of chymase in dermatitis (n=3 to4). Further, histamine, an inflammation mediator of mast cells, wassimilarly administered intradermally and the time-course was comparedwith the case of administration of human chymase. The histamine(Sigma-Aldrich) was injected by dissolving in saline (0.25 mg/ml).

As shown in FIG. 3A, by administering human chymase (2.0 μg/ear) to theears of mice, a biphasic edematous reaction resembling the allergic skinreaction shown in the case of Example 2 was induced. That is, the firstskin reaction was immediately induced after administration of chymaseand reached a peak after 30 minutes to 1 hour. Further, the second skinreaction peaked after 6 hours and continued for at least 24 hours. Onthe other hand, an immediate edematous reaction was induced even wheninoculating histamine, but this skin reaction completely disappeared 20hours after the inoculation in contrast to the case of inoculation ofchymase (FIG. 3B). The analysis of the dose-dependency in thechymase-induced dermatitis revealed that early-phase reaction (after 1hour) is dose-dependent and that the maximum reaction is observed at 2.0μg/ear amount the dose used in the experiment (FIG. 4A). On the otherhand, in the second (after 16 hours) reaction, while the reactions at0.5 μg/ear and 1.0 μg/ear were about the same in level, the maximumresponse was obtained at 2.0 μg/ear in the same way as in the firstreaction (FIG. 4B).

As shown above, it was shown that intradermal administration of humanchymase in mice induces dermatitis, and that its time-course resemblesthat of antigen-induced biphasic dermatitis, an acute model ofdermatitis, showing the involvement of chymase in biphasic dermatitis.

Example 5 Involvement of Chymase Activity in Dermatitis Induced bySingle Administration of Chymase

The ability of heat-treated human chymase to induced dermatitis wasinvestigated for the purpose of studying whether the enzymatic activityof chymase is involved in dermatitis induced by human chymase shown. Thehuman chymase was inactivated by incubating a 0.1 mg/ml human chymasesolution at 50° C. for 2 hours, then boiling it at 100° C. for 5minutes. This inactivated human chymase (2.0 μg/ear) was administered tothe ears of mice by the method described in Example 4, and thedermatitis was evaluated 1 hour after the administration.

As a result of the heat treatment of the human chymase, the edemareaction induced by the human chymase completely disappeared (p<0.01 vs.untreated chymase administration group, Student's t-test, N=4) (FIG. 5).This result shows that chymase activity is essential for inducingdermatitis.

Example 6 Histological Analysis of Dermatitis Induced by SingleAdministration of Chymase

A pathohistological analysis of dermatitis induced by chymase wasconducted and a comparison was performed with the biphasic dermatitisshown in Example 2 for the purpose of investigating in further detailthe involvement of chymase in biphasic dermatitis. These types ofdermatitis were induced in accordance with the methods described inExample 2 and Example 4 (dosage of human chymase was 2.0 μg/ear). Theears were fixed in formalin and paraffin sections were prepared inaccordance with an ordinary method 1 hour and 24 hours afteradministration in both models. The sections were stained withhematoxylin and eosin, then observed under-microscope and photographed.Further, sections of ears of normal BALB/c mice were used as negativecontrols.

In the section 1 hour after elicitation of chymase dermatitis (FIG. 6D),remarkable thickening was observed compared with the sections of ears ofnormal mice (FIG. 6A), but no difference could be observed between thetwo in respect to infiltration of leukocytes. As shown in FIG. 6E,however, remarkable cellular infiltration was observed in sections 24hours after chymase injection. The pattern of the histological changeshown by chymase dermatitis resembled that of the ascaris-inducedbiphasic dermatitis model (FIGS. 6B and 6C). That is, there wasremarkable thickening of the tissue at 1 hour, but cellular infiltrationwas observed only in sections after 24 hours. In summary, it was shownthat the dermatitis induced by chymase resembles the biphasic dermatitisinduced by an antigen even in a pathohistological analysis.

In the following Example 7 and Example 8, an analysis was conducted onthe mechanism of dermatitis induced by chymase to investigate the roleof chymase in biphasic dermatitis.

Example 7 Ability of Human Chymase to Induce Dermatitis in MastCell-Deficient Mice

Since it has been reported that chymase induces a degranulation in ratperitoneal mast cells (J. Immunol. 136, 3812, 1986), it was consideredpossible that dermatitis induced by chymase is induced through therelease of the inflammation mediator from the mast cells. Thus, next theinvolvement of mast cells in chymase-induced dermatitis was studiedusing mast-cell deficient mice (Blood 52, 447-425, 1978). Mastcell-deficient (WBB6F1-W/W^(v)) mice and their control mice (WBB6F1-+/+)were obtained from SLC Japan. Chymase dermatitis was induced byintradermal administration of human chymase (2.0 μg/ear) and the edemareaction was evaluated after 1 hour and 16 hours using the methoddescribed in Example 4.

As shown in FIGS. 7A and 7B, a skin reaction of a similar extent as thecontrol mice (WBB6F1-+/+) was observed even in mast cell deficient(WBB6F1-W/W^(v)) mice after 1 hour (FIG. 7A) and after 16 hours (FIG.7B). This result indicates that chymase induces a biphasic phase skinreaction regardless of the existence of mast cells.

Example 8 Ability of Human Chymase to Promote Migration ofPolymorphonuclear Leukocytes

In Example 6, it was shown that a remarkable infiltration of leukocytesis observed in late-phase reaction of human chymase-induced dermatitis.The effect of human chymase on the migration of polymorphonuclearleukocytes (PMN) in vitro was investigated for the purpose ofinvestigating the mechanism of chymase-induced leukocyte infiltration.PMN was isolated by adding a ⅕th volume of 6% dextran solution toheparinized whole blood from normal healthy subject and allowing it tostand at 37° C. for 1 hour, then layering the supernatent onFicoll-Paque (Amersham Pharmacia Biotech) and centrifuging it. Further,the migration of PMN was measured by using a 48-well chemotaxis chamber(NeuroProbe Co.) by the textbook method (Seibutsu Yakkagaku Jikken Koza(Biopharmacology Experiment Lectures) 12, 315, Hirokawa Shoten). Thatis, a medium containing human chymase (200 to 800 nM) or fMLP(N-formyl-L-methionyl-L-leucyl-L-phenylalanine, Sigma-Aldrich Co.) (10nM) was placed in the lower well of the chamber. The upper well and thelower well were separated by a polycarbonate filter (pore size 5 μm)(NeuroProbe Co.) PMN (1×10⁶/ml) was added to the upper well and culturedat 37° C. for 1 hour, then the filter was fixed, stained and the numberof cells in the membrane were counted under a microscope (400×)(Seibutsu Yakkagaku Jikken Koza (Biopharmacology Experiment Lectures)12, 315). For the cell staining, a hemacolor solution (Merck) was used.In this test, when investigating the effect of the chymase inhibitor, achymase inhibitor (Compound 18 or Compound 34) was dissolved in dimethylsulfoxide and added to the lower well of the chamber just before theaddition of the human chymase. The concentration of the compound wasadjusted so that the final concentration of the dimethyl sulfoxidebecame 1%.

As shown in FIG. 8A, human chymase exhibited an activity promotingmigration of human PMN in a concentration-dependent manner, with astatistical significance observed at ≧2400 nM (p<0.05, Dunnett's test).From the fact that human chymase exhibits activity promoting migrationto the same extent as 10 nM fMLP in a concentration of 200 to 400 nM, itis deduced to have an activity of about 1/30th that of fMLP. The actionof human chymase in promoting the migration of human PMN wassignificantly suppressed by chymase inhibitors, Compound 18 and Compound34 (FIG. 8B). These results suggest that chymase acts directly onpolymorphonuclear leukocytes to promote their migration, and show theinvolvement of enzymatic activity of chymase in that action.

Taken together, since chymase released by mast cells upon antigenstimuli induces biphasic skin reaction when injected intradermally tomice ear (Example 4 to 6), it is clear that chymase plays an importantrole in a skin reaction exhibiting biphasic reaction. The data ofExamples 7 and 8 suggest a mechanism of involvement of chymase inbiphasic skin reaction. In addition, it was also shown that a chymaseinhibitor suppresses ascaris-induced dermatitis, a biphasic skinreaction (Example 2 and Example 3).

Next, a mouse dermatitis model induced by repeated application of haptenwas analyzed as a model of dermatitis induced by repeated exposure to anantigen and the effects of a chymase inhibitor in this model is shown.

Example 9 Time-Course of Increase in Ear Thickness in Mouse DermatitisModel Induced by Repeated Application of Hapten

Dermatitis was induced in accordance with an already reported method (J.Pharmacol. Exp. Ther. 283, 321, 1997) using dinitrofluorobenzene (DNFB)as hapten. That is, the right ears of eight-week old female C3H/HeN mice(Nippon Clare) (n=7) were painted with 0.15% DNFB (25 μl) dissolved inan acetone/olive oil (3:1) every seven days six times. When applying thehapten each time, the ear thickness was measured by a micrometer(Digimatic Indicator, Mitsutoyo Co.) immediately before the applicationand 1 hour, 6 hours, 24 hours, and 48 hours after the application so asto find the amount of increase from the ear thickness before the firstapplication of hapten. Further, a group similarly treated with anacetone/olive oil solution (3:1) not containing DNFB was used as acontrol. Further, in a test separate from the above, the ears were cutoff immediately before the third application of hapten, the chymase-likeactivity of the skin was measured in accordance with an already reportedmethod (n=3), and this was compared with the activity of mice treatedwith an acetone/olive oil solution (3:1) not containing DNFB (n=2).

A transient skin reaction was induced by the application of DNFB. Thistransient skin reaction gradually became larger with each instance ofapplication (FIG. 9). By the repeated application of DNFB, in additionto an increase in the response to DNFB, the ear thickness beforeapplication (baseline value) gradually increased. For example, at thefifth week after the start of application, the thickness of the earimmediately before application increased by about 240 μm compared withthe ear before the first application (FIG. 9). In the control grouptreated with only a solvent not containing DNFB, almost no thickening ofthe ear was detected at any point of time. The chymase activity of theear immediately before the third application was significantly higher ascompared with the control group treated with only a solvent notcontaining DNFB (p<0.05, Student's t-test) (FIG. 10). In summary, it wasshown that repeated application of hapten DNFB to the ears of miceinduces a sustained ear thickness, in addition to an increase in thechymase activity and a remarkable transient skin reaction.

Example 10 Effect of Chymase Inhibitor in Mouse Dermatitis Model Inducedby Repeated Application of Hapten

The effect of a test substance on dermatitis was investigated byinducing dermatitis in accordance with the method described in Example 9(n=7) and measuring the ear thickness in the same way as in Example 9.As the test substance, three chymase inhibitors (Compound 18, Compound34, and Compound 35) were used, while as the control, the steroidprednisolone (Nakarai Tesc Inc.) was used. Further, the test substancewas suspended in saline containing 0.5% hydroxypropyl cellulose in thesame way as in Example 3 (HPC/saline) and intraperitoneally administeredin dosages of 10 mg/kg or 50 mg/kg once a day for five consecutive daysa week until the end of the test; the first administration was performedimmediately before the start of the hapten-application. Further, a groupsimilarly treated with DNFB and administered HPC/saline instead of thetest substance was used as a control group.

Prednisolone suppressed dermatitis in this model in a dose-dependentmanner (FIG. 11A). On the other hand, a chymase inhibitor alsoremarkably suppressed the transient skin reaction induced by theapplication of hapten, particularly after the fourth week (3 weeks afterinitial application of hapten) (FIG. 11B-D). For example, Compound 35significantly suppressed the increase in ear thickness in the fourth tosixth weeks (3 weeks initial application of hapten, and thereafter) at50 mg/kg 1 hour, 6 hours, 24 hours, and 48 hours after haptenapplication (p<0.05, Dunnett's test). Compound 34 also significantlysuppressed the skin reaction 1 to 48 hours after the application afterthe fifth week (4 weeks after initial application of hapten, andthereafter) at 50 mg/kg and exhibited a significant suppressive effect1, 24, and 48 hours after the fifth week (after the fifth application ofhapten) and 1 hour after the sixth week (after sixth application ofhapten) in an amount of 10 mg/kg. Further, Compound 18 significantlysuppressed the reaction 1 to 24 hours after the application in thefourth and fifth weeks (after fourth application and fifth applicationof hapten) and the reaction 1 to 48 hours in the sixth week (after sixthapplication of hapten) at 50 mg/kg and exhibited a significant effect 1to 48 hours after the application in the sixth week (after sixthapplication of hapten) even at 10 mg/kg.

These results show that chymase inhibitor suppresses edema in mousedermatitis model induced by repeated application of hapten.

Example 11 Effect of Chymase Inhibitor on Increase in Eosinophils ofSkin in Mouse Dermatitis Model Induced by Repeated Application of Hapten

The effect of a chymase inhibitor on the infiltration of eosinophils ofthe skin in the test of Example 10 was studied. That is, 48 hours afterthe sixth application of hapten, the ears of the mice were fixed withformalin and paraffin sections prepared in accordance with an ordinarymethod. The sections were stained with Fast Green (Fluka), known tospecifically stain eosinophils (Current Protocol in Immunology, WileyInterscience). That is, the deparaffined sections were fixed with 100%methanol for 1 minute, then stained for 30 minutes by 0.2% Fast Greendissolved in 70% ethanol. The eosinophils were measured by randomlyselecting 10 fields (X400) under a microscope and counting the number ofcells per area using an ocular grid. As the chymase inhibitor, theCompound 18 and Compound 34 were used and administered by the methoddescribed in Example 10. Further, a group treated with an acetone/oliveoil solution (3:1) not containing DNFB was used as the non-inducedcontrol group, while a group treated with DNFB and administeredHPC/saline instead of the test substance was used as the control for thegroup administered the chymase inhibitor.

As shown in FIG. 12, the number of eosinophils remarkably increased bythe repeated application of hapten (about 22 times that of the controlgroup, p<0.01, Student's t-test). As a result of administration of achymase inhibitor (Compound 18), the increase in the eosinophils wassignificantly suppressed in a dose-dependent manner. That is, the ratesof suppression at 10 mg/kg and 50 mg/kg were 37.1% and 60.5%,respectively. For the Compound 34 as well, while no statisticallysignificant difference was recognized, a trend toward suppressiondependent on the dosage was shown. These results suggest that chymaseinhibitor suppresses the increase in eosinophils of the skin in mousedermatitis model induced by repeated application of hapten.

Example 12 Effect of Chymase Inhibitor on Increase in Number of MastCells of Skin in Mouse Dermatitis Model Induced by Repeated Applicationof Hapten

The effect of a chymase inhibitor on the increase in mast cells of skinin the test of Example 10 was studied. In the same way as Example 11,the ears of the mice were fixed with formalin and paraffin sectionsprepared by an ordinary method 48 hours after the sixth application ofhapten. The mast cells of the sections were stained by the ToluidineBlue method, then the number of mast cells were counted under amicroscope (X400) for 10 fields per section. The density of mast cellsof the skin was measured in accordance with the method of Kitagaki etal. (J. Invest. Dermatol. 105, 749, 1995). As the chymase inhibitor, usewas made of Compound 18 and Compound 34. These were administered by themethod described in Example 10. Further, a group treated with anacetone/olive oil solution (3:1) not containing DNFB was used as thenon-induced control group, while a group treated with DNFB andadministered HPC/saline instead of the test substance was used as thecontrol for the group administered the chymase inhibitor.

As shown in FIG. 13, the density of mast cells of the skin significantlyincreased by the application of hapten (about 2.5 times that of thenon-induced group), but as a result of administration of a chymaseinhibitor (Compound 18 and Compound 34), the increase in the density ofmast cells was significantly suppressed by both the compounds in alldosages. The rate of suppression by Compound 18 was about 57% at 10mg/kg and about 64% at 50 mg/kg, while the rate of suppression byCompound 34 was about 37% at 10 mg/kg and about 51% at 50 mg/kg. FIG. 14shows representative micrographs. FIG. 14A shows the ear of anon-induced mouse, FIG. 14B shows the ear of a mouse treated with DNFBand administered HPC/saline (48 hours after the sixth application ofDNFB), and FIG. 14C shows the ear a mouse treated with DNFB andadministered Compound 34 (48 hours after the sixth application of DNFB).These results suggest that chymase inhibitor suppresses the increase inthe number of mast cells of the skin in mouse dermatitis model inducedby repeated application of hapten.

Example 13 Ability of Repeated Administration of Human Chymase to InduceDermatitis

The role of chymase in dermatitis induced by repeated exposure to anallergen was investigated by repeatedly administering intradermallyhuman chymase to the ear of mice. The human chymase was administeredonce a week (2.0 μg/ear/shot) in accordance with the method described inExample 4. The thickness of the ear was measured by a micrometer(Digimatic Indicator, Mitsutoyo Co.) immediately before eachadministration of chymase and 1 hour, 6 hours, 24 hours, and 48 hoursafter the administration to find the increase from the ear thicknessbefore the first administration of chymase. Further, heat-treatedchymase was prepared by the method described in Example 5, and itseffect was also studied at the same time.

A transient skin reaction was induced by intradermal administration ofhuman chymase to the ears of the mice. The reactivity with respect tochymase was about the same among the first to third administrations ofchymase, but was amplified by the fourth to fifth administrations (FIG.15). No transient reaction was observed when the inactivated chymase wasadministered repeatedly. Further, no skin reaction at all was observedwhen repeatedly administering this inactivated chymase (FIG. 15). Thisshows that the skin reaction observed was due to the enzymatic activityof chymase and that the amplification of the skin reaction seen whenadministering chymase four to five times is not due to theadministration of foreign protein to the mice.

Example 14 Effect of Repeated Administration of Human Chymase on Numberof Eosinophils of Skin

Human chymase was repeatedly administered to the ears of mice toinvestigate the change in the number of eosinophils of the skin. Humanchymase was repeatedly administered to the ears of mice in accordancewith the method described in Example 13. The number of eosinophils ofthe skin was measured by the method described in Example 11. Further, asa control, ear tissue repeatedly administered saline was used.

As shown in FIG. 16, in an ear 24 hours after single administration of2.0 μg of human chymase, there was an approximately 7.6 fold increase inthe number of eosinophils compared with an ear administered saline(p<0.01, Student's t-test). When human chymase was administered furtherat 1 week intervals four times in total and the number of eosinophilswas measured 1 week after each administration, the number of eosinophilsfurther increased to about 21-fold the group administered saline(p<0.01, Student's t-test). These results indicate the possibility thatchymase increases the number of eosinophils and that the rate of theincrease is dependent on the frequency of the exposure to chymase.

Example 15 Effect of Repeated Administration of Human Chymase on Numberof Mast Cells of Skin

Human chymase was repeatedly administered intradermally to the ears ofmice to investigate the change in the number of mast cells of the skin.Human chymase was administered repeatedly in accordance with the methoddescribed in Example 13 and the change in the mast cells of the skin wasstudied 1 week after administration of the chymase by the methoddescribed in Example 12 or the method of measuring the histamine contentof the skin. The skin histamine content was measured by cutting off theear, then homogenizing it in a 20 mM Tris-HCl buffer (pH7.5),centrifuging the extract (10,000 rpm, 10 minutes), then assaying theamount of histamine in the supernatent using an ELISA kit (MedicalBiological Laboratories). Further, as the control, use was made of eartissue repeatedly administered saline.

The change in the number of mast cells of the skin was observed inaccordance with the method described in Example 12, whereupon a tendencyfor an increase in the number of mast cells by the administration ofhuman chymase was seen. The increase in the number of cells however wasnot remarkable. Thus, the histamine content of the skin, an indicator ofthe number of mast cells of tissue, was measured for the purpose of moreobjectively or quantitatively evaluating the increase in the number ofmast cells of the skin. As a result, as shown in FIG. 17, the histaminecontent of the skin was a significantly higher than the groupadministered saline a week after the fourth administration of humanchymase. From these results, the existence of a mechanism where thechymase derived from mast cells acts on the mast cells in a positivefeedback manner is suggested.

Example 16 Effect of Administration of Human Chymase on Stem CellFactors (SCF)

The expression of stem cell factors (SCF), known as a factor fordifferentiation and proliferation of mast cells (Blood 90, 1345, 1997),was analyzed by the immunohistochemical method for the purpose ofclarifying the mechanism of action of the increase in the number of mastcells of the skin when administering human chymase. Human chymase (2.0μg/ear) was administered to mouse ears by the method described inExample 4. The ears were harvested after 1 hour, after 6 hours, andafter 24 hours and frozen tissue sections of 5 μm were prepared by anordinary method. As a control, sections of ears of normal mice wereused. In the immunohistochemical studies, anti-mouse SCF goat IgG (madeby R&D Systems) was used as the primary antibody for detection by a PAPkit (DAKO Co.) Note that as a negative control, normal goat IgG (VectorLaboratories Co.) was used instead of an SCF antibody. Further, afterimmunostaining, nucleus was stained using Methyl Green (Wako PureChemicals) in accordance with an ordinary method (Sensyokuho no Subete(Everything About Dyeing), Ishiyaku Shuppan).

In normal mouse ears, a strong immunostaining was observed around thecorneal layer of the epidermis (FIG. 18A). On the other hand, noimmunostaining at all was seen when using normal goat IgG instead ofanti-SCF antibody as the primary antibody. This shows that theimmunostaining in the corneal layer is specific. On the other hand, inthe ears of mice administered chymase, the staining around the corneallayer becomes weaker in a time-dependent manner. After 24 hours, almostno formation of color is seen in the ears (FIG. 18B).

Example 17 Effect of Human Chymase on Expression of SCF in HumanKeratinocytes

The effect of human chymase on the expression of SCF in human normalkeratinocytes in vitro was studied. Human normal keratinocytes wereobtained from Cell Applications Co. The cultured keratinocytes wereharvested by an enzyme-free cell dissociation buffer (GIBCO BRL Co.),washed three times by PBS, then adjusted to a cell concentration of10⁶/50 μl, added with human chymase, and allowed to react at 37° C. for10 minutes. The chymase reaction was stopped by the addition of fetalcalf serum (FCS) to give a final concentration of 10%, then the cellswere removed by centrifugation. The SCF in the supernatent was assayedby an ELISA kit (PeproTech Co.) The cytotoxicity of chymase to thekeratinocytes was investigated using as an indicator the release oflactate dehydrogenase (LDH) using a cytotoxicity detection kit (made byRoche Molecular Biochemicals Co.)

As shown in FIG. 19, the release of SCF was promoted in aconcentration-dependent manner by incubating human keratinocytes in thepresence of human chymase for 10 minutes. The amount of release of SCFwhen incubating it in the absence of human chymase for 10 minutes wasabout the same as when not incubating it. On the other hand, the releaseof LDH in the supernatent did not change all under these conditions. Itwas confirmed that the release of SCF due to human chymase was not bycell damage. Above, it was shown in vitro that human chymase acts onmembrane-bound SCF of human keratinocytes and causes the release of freeSCF.

SCF in composed of two types of molecules of SCF²⁴⁸ and SCF²²⁰ that aregenerated by the differences in splicing (Blood 90, 1345, 1997). SCF²⁴⁶is first synthesized as a cell membrane protein, then processed by somesort of protease to become free SCF which then is released from thecells. On the other hand, SCF²²⁰ does not have any site digested by anenzyme, so functions only as a membrane protein (Blood 90, 1345, 1997).Longley et al. have provided data that the SCF of the skin of healthysubjects is expressed mainly on the cell membranes of keratinocytes ofthe epidermis, but that in dermatitis accompanied with an increase inskin mast cells, the expression of SCF on the cells is no longerobserved and that SCF is detected in the dermis and the intercellularspaces of the keratinocytes (N. Engl. J. Med. 328, 1302, 1993). Further,in transgenic mice made to excessively express both of SCF²²⁰ andSCF²⁴⁸, an increase in the number of skin mast cells is observed, butthis phenomenon is not seen in transgenic mice made to excessivelyexpress only SCF²²⁰ (J. Exp. Med. 187, 1565, 1998). These findingssuggest that the patho physiological roles differ between membrane-boundtype and free SCF and in particular that free SCF is closely related tothe increase in number of mast cells of the skin. In fact, it is knownthat administration of free SCF to the human skin causes an increase inthe number of mast cells of the skin (J. Exp. Med. 183, 2681, 1996).

Longley et al. further report that human chymase cleaves membranebinding SCF and converts it to free SCF (Proc. Natl. Acad. Sci. USA, 94,9017; 1997). The findings obtained from Example 16 are completely novelfindings proving the data of Longley et al. in vivo. The findings ofExample 17 are initial data shown using human cells. Further, thefindings of Examples 16 and 17 can be said to be data explaining theincrease in skin mast cells induced by administration of chymase shownin Example 15 and the mechanism of action in suppressing the increase inskin mast cells by a chymase inhibitor shown in Example 12.

From the fact that skin reaction that is induced by administratingartificially from the outside chymase released by mast cells uponantigen stimuli is amplified along with the increase in the number ofthe repeated administration, (Example 13), it is clear that chymaseplays an important role in dermatitis induced by repeated exposure to anantigen. Further, from the fact that a chymase inhibitor suppresses theincrease in eosinophills or mast cells in skin in dermatitis induced byrepeated application of hapten (Examples 11 and 12) and that theadministration of chymase causes an increase in eosinophils or mastcells (Examples 14 to 15), it is clear that chymase controls the numberof eosinophils or mast cells playing an important role in allergicreactions. Further, it was shown that a chymase inhibitor improves thecondition of dermatitis in the dermatitis model induced by repeatedapplication of hapten, which is a model of dermatitis induced byrepeated exposure to an antigen (Examples 9 and 10).

Next, the effect of a chymase inhibitor on natural onset dermatitis(NC/Nga) mice as a second model of dermatitis induced by repeatedexposure to an antigen will be shown.

Example 18 Effect of Chymase Inhibitor on Natural Onset DermatitisNC/Nga) Mice

NC/Nga mice were bred and raised in accordance with the methodsdescribed in previous reports (Progress in Medicine 19, 1201, 1999).Specifically, 5-week old NC/Nga mice obtained from Charles River Japanand raised under a specific pathogen free (SPF) environment were mixedwith NC/Nga mice bred by the Faculty of Applied Biological Science ofHiroshima University starting from 6 weeks of age, kept until 15 weeksof age in a normal non-SPF environment, then used for the tests. Thetests were conducted under non-SPF, conventional environment. The testsubstance (Compound 18) was mixed with the drinking water andadministered for 35 consecutive days at 150 mg/kg/day (n=7), then anevaluation was conducted in accordance with the method described inprevious reports (Progress in Medicine 19, 1201, 1999). That is, first,35 days after the start of administration, the five items of (1)scratching behavior, (2) edema, (3) erythema and hemorrhaging, (4)depilation and ulceration, and (5) dryness were scored as 0 to 2 and thetotal found to judge the outer appearance of the skin. Next, the earsand skin of the back were fixed with formalin, embedded in paraffin, andhistologically analyzed by staining the ear specimens by the hematoxylinand Eosin method, Toluidine Blue method, or Congo Red method (StainTechnol. 56, 323, 1981) and by staining the back skin specimens by theToluidine Blue method or Congo Red method. Sections of the ear stainedwith Hematoxylin and Eosin were evaluated by judging the change intissue in four scales of 0 to 3 for three items of (1) thickness ofepidermis, (2) thickness of dermis, and (3) cellular infiltration andexpressing as the total score. Further, the specimens stained withToluidine Blue and stained with Congo Red were used for counting themast cells and eosinophils. Specifically, the eosinophils of the backskin were counted in a X400 field of a microscope, while the other cellswere counted in a total of five X200 fields and the totals expressed.

As shown in FIGS. 20A and 20B, at the start of the test (15 weeks age),no difference could be seen in the scores of the skin conditions betweenthe control group and the chymase inhibitor (Compound 18) administeredgroup (FIG. 20A), but 35 days after administration of the chymaseinhibitor, the dermatitis score decreased significantly compared withthe control group (p<0.05, Mann-Whitney test) (FIG. 20B). Similarly, thehistological score of the ears also decreased significantly by theadministration of a chymase inhibitor (FIG. 21, p<0.05, Mann-Whitneytest). On the other hand, the mast cells of the ear skin (FIG. 22A) andback skin (FIG. 22B) were counted. As a result, a significantsuppressive effect was recognized by the administration of a chymaseinhibitor in both specimens. Further, it was shown that the numbers ofeosinophils of the ear skin (FIG. 23A) and back skin (FIG. 23B) weresignificantly suppressed by a chymase inhibitor.

Above, in a natural onset atopic dermatitis model where onset isconsidered to occur due to repeated exposure to an antigen in the airunder a non-SPF environment, a chymase inhibitor improves the outerappearance of the skin and the histological change of the skin andsuppresses the increase in mast-cells and infiltration of eosinophils,so the usefulness of a chymase inhibitor against dermatitis induced byrepeated exposure to an antigen is demonstrated.

Formulation Example 1 Production of Tablets

100.0 g of Compound 1 was mixed with 22.5 g of microcrystallinecellulose and 2.5 g of magnesium stearate and then tabletized by asingle-action type tabletizing machine to produce tablets of a diameterof 9 mm and a weight of 250 mg each containing 200 mg of Compound 1.

Formulation Example 2 Production of Granules

30 g of Compound 1 was mixed well with 265 g of lactose and 5 g ofmagnesium stearate. The mixture was press molded, then pulverized,granulated, and sieved to obtain excellent 10% granules of 20 to 50mesh.

Formulation Example 3 Production of Rectal Suppositoru

Vitepsol H-15 (Dynamite Nobel Co.) was warmed to melt. To this was addedCompound 1 to a concentration of 12.5 mg/ml. This was homogeneouslymixed, then was added in 2 ml amounts to rectal suppository mold andcooled to obtain rectal suppositories each containing 25 mg of Compound1.

INDUSTRIAL APPLICABILITY

According to the present invention, a chymase inhibitor alleviates abiphasic skin inflammation reaction or its late-phase reaction and iseffective against skin thickening of dermatitis induced by repeatedapplication of hapten, one of the animal disease models of atopicdermatitis, and can effectively prevent and/or treat conditions ofdermatitis exhibiting a biphasic inflammation reaction or dermatitisinduced by repeated exposure to an antigen.

1-16. (canceled)
 17. A method for prevention or treatment of dermatitisexhibiting a biphasic skin reaction comprising administering to apatient in need of such treatment a chymase inhibitor in an amounteffective for alleviating late-phase reaction.
 18. A method according toclaim 17, wherein the dermatitis exhibiting a biphasic skin reaction isatopic dermatitis.
 19. A method for alleviation of late-phase reactionof dermatitis exhibiting biphasic skin reaction comprising administeringa chymase inhibitor to a patient in need of such treatment in an amounteffective for alleviating late-phase reaction of dermatitis exhibitingbiphasic skin reaction.
 20. A method according to claim 19, wherein saiddermatitis exhibiting a biphasic skin reaction is atopic dermatitis. 21.A method for the prevention or treatment of dermatitis induced byrepeated exposure to an antigen comprising administering a chymaseinhibitor to a patient in need of such treatment in an amount effectivefor prevention or treatment of said dermatitis.
 22. A method accordingto claim 21, wherein said dermatitis induced by repeated exposure to anantigen is atopic dermatitis.